Susanna Pietropaolo
email: susanna.pietropaolo@u-bordeaux.fr

Giovanni Marsicano

The role of the endocannabinoid system as a therapeutic target for autism spectrum disorder: Lessons from behavioral studies on mouse models

Keywords:

Recent years have seen an impressive amount of research devoted to understanding the etiopathology of Autism Spectrum Disorder (ASD) and developing therapies for this syndrome. Because of the lack of biomarkers of ASD, this work has been largely based on the behavioral characterization of rodent models, based on a multitude of genetic and environmental manipulations. Here we highlight how the endocannabinoid system (ECS) has recently emerged within this context of mouse behavioral studies as an etiopathological factor in ASD and a valid potential therapeutic target. We summarize the most recent results showing alterations of the ECS in rodent models of ASD, and demonstrating ASD-like behaviors in mice with altered ECS, induced either by genetic or pharmacological manipulations. We also give a critical overview of the most relevant advances in designing treatments and novel mouse models for ASD targeting the ECS, highlighting the relevance of thorough and innovative behavioral approaches to investigate the mechanisms acting underneath the complex features of ASD.

1. Autism Spectrum Disorder (ASD) and its animal models: a matter of complexity 1.1. The complex nature and definition of ASD Autism Spectrum disorder (ASD) is probably among the most complex pathologies of the central nervous system, starting from its diagnostic definition [START_REF] Dover | How to diagnose autism[END_REF][START_REF] Klin | Affording autism an early brain development re-definition[END_REF]. The criteria for diagnosing autism, as the disorder was originally called, were firstly proposed by Kanner [START_REF] Kanner | Autistic disturbances of affective contact[END_REF], subsequently refined by Rutter [START_REF] Rutter | Concepts of autism: a review of research[END_REF]) and then reformulated several times in the Diagnostic and Statistical Manual of Mental Disorders (American psychiatric association, 2013). According to the original definitions, a child can be ascertained for autism when the onset of the disturbance, characterised by pervasive social impairment and deficits in language and/or communication, occurred before 30 months, in the absence of the delusions and hallucinations typically found in schizophrenic patients (American psychiatric association, 1980). The subsequent revisions of the DSM tended to isolate pervasive developmental disorders, highlighting that autistic symptomatology is instead not confined to early childhood, but persists afterwards (American psychiatric association, 1987). Overall, the basic diagnostic criteria of autism include the following triad: social dysfunction, qualitative deficits in ver-bal/nonverbal communication and a restricted range of interests associated with the occurrence of repetitive behaviors. These criteria were substantially maintained in the more recent editions of DSM, although the first and second domains tended to be merged into a more general field of social/communication deficits, thus eliminating the triad concept (American psychiatric association, 2013).

Introducing the concept of "spectrum" (American psychiatric association, 2000) has drastically changed our view of autistic pathology: ASD is now defined as a single disorder that includes pathologies that were previously considered separate, i.e., autism, Asperger's syndrome, childhood disintegrative disorder and pervasive developmental disorder not otherwise specified (American psychiatric association, 2013). The term "spectrum" also refers to the wide range of symptoms and degrees of severity characterizing ASD patients, underlining the huge variability affecting the expression of both core (i.e., the ancient autistic "triad") and additional symptoms [START_REF] Rapin | Autistic children: diagnosis and clinical features[END_REF]. This impressively high heterogeneity of ASD is therefore related to the fact that it has been somehow artificially defined as a single disorder, but it is in reality a combination of divergent disorders, in turn all having variable symptoms.

The complexity of modelling ASD in laboratory animals: a challenge for behavioral testing

The complexity of the nature of ASD and of its definition obviously complicates the designing of animal models of this disorder. The latter task is already made difficult by the unknown aetiology of the disorder, including complex multigenic and environmental factors, with specific contributions and underlying mechanisms that still remain undefined. Furthermore, the lack of biomarkers of autistic pathology directs most of the possible research towards the behavioral validation of its animal models. Assessing the face validity of animal models of ASD is also a challenging and complex task, because of the large variety of behavioral symptoms. While additional ASD-relevant symptoms such as anxiety, sensory abnormalities, hyper-activity, and cognitive alterations can be tested in mice with well-established and standardized behavioral protocols, most of the core autistic symptoms are instead not easily modelled in rodents [START_REF] Servadio | Modeling autism-relevant behavioral phenotypes in rats and mice: do' autistic' rodents exist?[END_REF]. The core autistic symptom of the occurrence of repetitive behaviors and insistence on the sameness has been evaluated in laboratory mice through indirect measures of behavioral flexibility in different types of mazes (i.e, deficits in spontaneous alternation in Y-or T-mazes, or in reversal learning or working memory in the water maze), or through the assessment of home-cage or novelty-induced behavioral stereotypies [e.g. excessive self-grooming or marble burying [START_REF] Moy | Development of a mouse test for repetitive, restricted behaviors: relevance to autism[END_REF]]. Novel protocols testing habit formation and insistence on sameness have been also developed in order to better model this behavioral ASD-like abnormality [START_REF] Amodeo | Differences in BTBR T+ tf/J and C57BL/6J mice on probabilistic reversal learning and stereotyped behaviors[END_REF][START_REF] Cupolillo | Autistic-like traits and cerebellar dysfunction in purkinje cell PTEN knock-out mice[END_REF][START_REF] Roullet | Mouse models of autism: testing hypotheses about molecular mechanisms[END_REF].

Modelling the ASD-like core social deficits in laboratory rodents is probably the most challenging task, since autism seems intrinsically related to social dysfunction. Furthermore, proposing relevant parallelisms between human and mouse social behavioral test represents quite a challenge, because of the longer preferential use of rats in studies on social behaviors [START_REF] Kondrakiewicz | Ecological validity of social interaction tests in rats and mice[END_REF]. Hence, a large body of recent research has focused on developing more appropriate methods to assess social ASD-like behaviors in mice. Social interest for example has been assessed in juvenile and adult mice using social preference tasks in the three chamber test [START_REF] Nadler | Automated apparatus for quantitation of social approach behaviors in mice[END_REF], or direct interaction tests, also evaluating juvenile play, affiliative behaviors, or aggression. Furthermore, communication has also been assessed in laboratory mice, as they emit ultrasonic vocalizations in social contexts at multiple stages of their life [START_REF] Wohr | Ultrasonic vocalizations in Shank mouse models for autism spectrum disorders: detailed spectrographic analyses and developmental profiles[END_REF][START_REF] Wohr | Behavioural methods used in rodent models of autism spectrum disorders: current standards and new developments[END_REF], i.e., during isolation on the first post-natal days [START_REF] Branchi | Ultrasonic vocalisation emitted by infant rodents: a tool for assessment of neurobehavioural development[END_REF][START_REF] D'amato | Pups call, mothers rush: does maternal responsiveness affect the amount of ultrasonic vocalizations in mouse pups?[END_REF] and during adult social interactions [START_REF] Holy | Ultrasonic songs of male mice[END_REF][START_REF] Moles | Ultrasonic vocalizations emitted during dyadic interactions in female mice: a possible index of sociability?[END_REF]. The analysis of all these mouse social behaviors has impressively evolved, leading to advanced tools for data analysis and to novel experimental protocols to assess complex social behaviors.

An example of a major effort to refine the analysis of mouse social behaviors comes from recent research proposing the automatized evaluation of social interactions in animal dyads and groups, using machine learning strategies [START_REF] De Chaumont | Real-time analysis of the behaviour of groups of mice via a depth-sensing camera and machine learning[END_REF][START_REF] Mathis | DeepLabCut: markerless pose estimation of user-defined body parts with deep learning[END_REF][START_REF] Nath | Using DeepLabCut for 3D markerless pose estimation across species and behaviors[END_REF]. These recent approaches try to eliminate the major limiting factors affecting the analysis of mouse social behaviors, i.e., its time-consuming and observer-dependent characteristics, often pushing away researchers from including the evaluation of social phenotypes in their batteries of tests for genetic mouse models. Because of the necessary expertise in programming and informatics, the extensive application of these advanced technological tools to the analysis of mouse social behavior still requires some time, but it will probably become accessible to a larger audience very soon. Obviously, their application could reveal interesting outcomes and general advantages in rats too, to be adopted in future studies on social behaviors.

Among the most impressive advances in data analysis are probably those addressing mouse ultrasonic communication, since the phenomenon started being studied with basic bat-detectors and simple quantifi-cation of the calls and then moved to sensitive microphones and sophisticated qualitative assessment of ultrasonic spectrograms [START_REF] Ferhat | Recording mouse ultrasonic vocalizations to evaluate social communication[END_REF]. These advances allowed the researchers to identify different type of calls and syllables within the repertoire of an adult mouse, and to study their modulation by the social context and other parameters [START_REF] Hanson | Female presence and estrous state influence mouse ultrasonic courtship vocalizations[END_REF][START_REF] Kikusui | Testosterone increases the emission of ultrasonic vocalizations with different acoustic characteristics in mice[END_REF]Matsumoto andOkanoya, 2016, 2018). These advances have so far concerned mostly the analysis of ultrasonic communication in mice, where they may specifically compensate the absence of frequency-specific calls with different affective value that are instead present in rats (as described in Section 1.3). This detailed qualitative analysis can also be applied during the first post-natal days, and it has allowed identifying alterations in the developmental ultrasonic profile of genetic models of ASD [START_REF] Agarwalla | Male-specific alterations in structure of isolation call sequences of mouse pups with 16p11.2 deletion[END_REF][START_REF] Fyke | Communication and social interaction in the cannabinoid-type 1 receptor null mouse: implications for autism spectrum disorder[END_REF][START_REF] Lai | Temporal and spectral differences in the ultrasonic vocalizations of fragile X knock out mice during postnatal development[END_REF][START_REF] Roy | Comprehensive analysis of ultrasonic vocalizations in a mouse model of fragile X syndrome reveals limited, call type specific deficits[END_REF]. The application of these novel analytical tools to genetic mouse models of ASD has indeed dramatically improved our ability to assess their face validity, often allowing subtle communicative alterations to emerge at different ages.

Finally, the evaluation of complex social behaviors in laboratory mice have benefitted from the recent designing of sophisticated experimental protocols e.g., targeting the social transmission of emotions and empathy [START_REF] Jeon | A mouse model for observational fear learning and the empathetic response[END_REF][START_REF] Jordan | Mice, pain, and empathy[END_REF][START_REF] Lecker | Physical contact promotes the development of emotional contagion between mice[END_REF]. Knowing the affective state of others is a critical factor that can drive social interactions, and it is directly linked to ASD since an inability to decipher another's affective state is indeed a core autistic feature. Recent elegant studies have demonstrated the social transmission of affective states in laboratory mice by direct social interaction between an observer with no aversive experience and a demonstrator having experienced electric shocks: the enhanced stress levels and fear responses detected in the observer represent an index of the social transmission of a negative affective state [START_REF] Sterley | Social transmission and buffering of synaptic changes after stress[END_REF][START_REF] Sterley | Social communication of affective states[END_REF]. The ability of transmitting to cagemates a painful experience has also been evaluated in mice, through the development of a formalin test for emotional contagion [START_REF] Laviola | Low empathy-like behaviour in male mice associates with impaired sociability, emotional memory, physiological stress reactivity and variations in neurobiological regulations[END_REF]. Other authors have instead used the restrain stress protocol to show that sharing the stress with cage mates can reduced corticosterone levels of laboratory mice, while experiencing the stress alone among cage mates has the opposite effect, suggesting evidence of empathy and reversed empathy in laboratory mice [START_REF] Watanabe | Empathy and reversed empathy of stress in mice[END_REF].

Advantages and limitations of mouse versus rat studies on ASD

As mentioned above, social behavior has been by tradition studied preferentially in laboratory rats, somehow supporting the idea of the mouse as a "less social" species, mostly affected by territoriality and less involved in complex social interactions and behaviors. This ancient idea has been largely overcome in the recent years, mostly because of the clear invaluable advantages of studying social behavior and its dysfunction in laboratory mice. The major advantage of mouse versus rat studies on social behavior lies in the simplicity of manipulating the mouse genome, resulting in the availability of a myriad of inbred mouse strains and of genetically modified mouse lines, offering the unique possibility of understanding the genetic substrates of most basic aspects of social interactions.

Laboratory mice have become therefore an invaluable tool to investigate the molecular mechanisms underlying several social behaviors, also at early stages. Juvenile mice for instance are known to express a robust motivation to approach conspecifics of the same age, and play behavior is known to be not exclusive of young rats [START_REF] Terranova | Ontogeny of amicable social behavior in the mouse: gender differences and ongoing isolation outcomes[END_REF]. Genetic studies in juvenile mice [e.g., (Panksepp et al., 2007)] can therefore provide promising targets for intervention with high predictive validity, having an obvious special relevance for neurodevelopmental disorders in general and ASD in particular. The rapid advancing of gene targeting techniques also offers to the researchers novel unique tools to further dissect the molecular mechanisms underlying complex social behaviors. This is the case of social transmission of affective states, that has been evaluated in genetically modified mice (see also Section 1.2), allowing to shed light on the precise neuronal circuits involved in this complex social interaction [START_REF] Sterley | Social transmission and buffering of synaptic changes after stress[END_REF][START_REF] Sterley | Social communication of affective states[END_REF]. Recently, it has been shown in mice that the control of behavioral responses during social interaction essays can be disentangled in its finer components, i.e., in terms of the specificity of the cellular populations (e.g., astroglial versus neuronal cells) and subcellular components (e.g., mitochondrial versus plasmatic receptors) involved in brain metabolic functions [START_REF] Jimenez-Blasco | Glucose metabolism links astroglial mitochondria to cannabinoid effects[END_REF]. Such a finely-tuned control of genetic manipulation of social functions in mice opens a brand new avenue of research perspectives that would be more difficult to be realized in laboratory rats, at least in terms of rapidity and precision of targeting.

Despite the clear advantages of using laboratory mice for studying social interactions and ASD, some aspects of social behaviors can still be investigated exclusively in rats. For example, although social play is studied in laboratory mice, rough-and-tumble play is uncommon in the mouse species (Panksepp and Lahvis, 2007), in contrast to rats where it constitutes a major behavioral component. Similarly, mice use ultrasonic communication during their social interactions, with levels of complexity that become increasingly evident, including the production of ultrasonic calls of different types according to the social context, as we described in Section 1.2. Nonetheless, the use of ultrasonic calls with different affective value has been so far demonstrated only in rats, since positive 50 kHz calls (also known as the rat laughter) and 22 kHz alarm calls have not been detected in mice so far [START_REF] Knutson | Ultrasonic vocalizations as indices of affective states in rats[END_REF][START_REF] Portfors | Types and functions of ultrasonic vocalizations in laboratory rats and mice[END_REF][START_REF] Wohr | Affective communication in rodents: ultrasonic vocalizations as a tool for research on emotion and motivation[END_REF]. This represents an unique advantage of the rat species to study affective communication of positive/reinforcing experiences, allowing to study the brain circuits and mechanisms underlying this important social function [START_REF] Burgdorf | Frequency-modulated 50 kHz ultrasonic vocalizations: a tool for uncovering the molecular substrates of positive affect[END_REF][START_REF] Burgdorf | Neurobiology of 50-kHz ultrasonic vocalizations in rats: electrode mapping, lesion, and pharmacology studies[END_REF], as well as the evolutionary role of communicative ways of sharing of positive versus negative feelings [START_REF] Panksepp | Laughing" rats and the evolutionary antecedents of human joy?[END_REF].

How do we define behaviorally "valid" mouse models for ASD?

To face the challenges of basic research on ASD, a promising approach seems to consist in modeling single symptoms, rather than the whole disorder, giving up any attempt of a unitary explanation of ASD, as suggested by some authors [START_REF] Happe | Time to give up on a single explanation for autism[END_REF]. The definition of the precise criteria that a valid model for ASD should fulfill is still the subject of an intense debate [START_REF] Chadman | Do animal models hold value in Autism spectrum disorder (ASD) drug discovery?[END_REF][START_REF] Kazdoba | Behavioral phenotypes of genetic mouse models of autism[END_REF][START_REF] Servadio | Modeling autism-relevant behavioral phenotypes in rats and mice: do' autistic' rodents exist?[END_REF]. An ideal animal model would be able to mimic all core behavioral autistic features in association with some of the secondary ones. Nonetheless, the behavioral validation of animal models for ASD is most often focused on the social domain only [START_REF] Crawley | Designing mouse behavioral tasks relevant to autistic-like behaviors[END_REF][START_REF] Crawley | Mouse behavioral assays relevant to the symptoms of autism[END_REF][START_REF] Moy | Mouse models of autism spectrum disorders: the challenge for behavioral genetics[END_REF][START_REF] Silverman | Behavioural phenotyping assays for mouse models of autism[END_REF], as evident also from the data summaries illustrated here in Tables 2 and3. Most reasonably, a rodent model should be considered as valid if it presents alterations in both core domains affected in ASD, i.e., social interaction/ communication, and flexible/repetitive behaviors [START_REF] Crawley | Designing mouse behavioral tasks relevant to autistic-like behaviors[END_REF][START_REF] Crawley | Mouse behavioral assays relevant to the symptoms of autism[END_REF][START_REF] Wohr | Behavioural methods used in rodent models of autism spectrum disorders: current standards and new developments[END_REF]. The presence of additional behavioral abnormalities, such as hyper-activity, memory deficits or anxiety, may increase the validity of the model, but is not required [START_REF] Kazdoba | Behavioral phenotypes of genetic mouse models of autism[END_REF][START_REF] Servadio | Modeling autism-relevant behavioral phenotypes in rats and mice: do' autistic' rodents exist?[END_REF].

As already reviewed in several articles [e.g., [START_REF] Banerjee | Genetic aspects of autism spectrum disorders: insights from animal models[END_REF][START_REF] Bey | Overview of mouse models of autism spectrum disorders[END_REF][START_REF] Ellegood | Behavioral and neuroanatomical phenotypes in mouse models of autism[END_REF][START_REF] Moy | Mouse models of autism spectrum disorders: the challenge for behavioral genetics[END_REF]], a large variety of mouse models for ASD have been proposed in the last decades, based on very different approaches. One strategy consists of identifying mouse strains that spontaneously present some major ASDlike symptoms, such as low sociability, poor communication abilities, and high levels of inflexible behaviors. The best-known example of this approach is the BTBR strain [START_REF] Mcfarlane | Autism-like behavioral phenotypes in BTBR T+tf/J mice[END_REF][START_REF] Meyza | The BTBR T+ tf/J mouse model for autism spectrum disorders-in search of biomarkers[END_REF], widely employed in ASD research and proposed as a tool to iden-tify novel genes involved in the control of social behaviors. This model is characterized by agenesis of the corpus callosum [START_REF] Wahlsten | Survey of 21 inbred mouse strains in two laboratories reveals that BTBR T/+ tf/tf has severely reduced hippocampal commissure and absent corpus callosum[END_REF], as well as by several behavioral abnormalities, including alterations in ultrasonic communication both at early post-natal and adult ages, reduced social interest in the three chamber test, high levels of self-grooming and deficits in cognitive flexibility [START_REF] Pearson | Motor and cognitive stereotypies in the BTBR T+tf/J mouse model of autism[END_REF][START_REF] Scattoni | Unusual repertoire of vocalizations in the BTBR T+tf/J mouse model of autism[END_REF][START_REF] Scattoni | Unusual repertoire of vocalizations in adult BTBR T+tf/J mice during three types of social encounters[END_REF][START_REF] Yang | Social approach behaviors are similar on conventional versus reverse lighting cycles, and in replications across cohorts, in BTBR T+ tf/J, C57BL/6J, and vasopressin receptor 1B mutant mice[END_REF]. The main disadvantages of this model are (i) the problem of identifying an appropriate control strain and (ii) the possibility that strain-specific phenotypes of emotionality may explain the supposed ASD-like behavioral profiles (Oddi et al., 2013b) and play as major confounding factors on the social abnormalities. These problems are somehow intrinsic of the strain approach and indeed affect also the equivalent rat models based on strain differences [e.g., [START_REF] Zhang-James | Autism-related behavioral phenotypes in an inbred rat substrain[END_REF]].

Other mouse models rely on the exposure (mostly prenatally) to adverse environmental factors [START_REF] Dufour-Rainfray | Fetal exposure to teratogens: evidence of genes involved in autism[END_REF][START_REF] Patterson | Maternal infection and immune involvement in autism[END_REF], such as certain teratogens/drugs and viral/bacterial infections, suspected to contribute to the etiology of ASD on the basis of epidemiological data (American College of and Gynecologists' Committee on Obstetric, 2021; [START_REF] Atladottir | Maternal infection requiring hospitalization during pregnancy and autism spectrum disorders[END_REF][START_REF] Davis | Autism and developmental abnormalities in children with perinatal cocaine exposure[END_REF][START_REF] Engman | Prenatal acquired cytomegalovirus infection should be considered in children with autism[END_REF][START_REF] Fluegge | Maternal infection during pregnancy, risk of offspring autism, and the role of bacterial denitrification[END_REF][START_REF] Mezzacappa | Risk for autism spectrum disorders according to period of prenatal antidepressant exposure: a systematic review and meta-analysis[END_REF][START_REF] Zerbo | Maternal infection during pregnancy and autism spectrum disorders[END_REF]. A well-known example of this approach is the valproic acid (VPA) model, developed mainly in rats [START_REF] Ingram | Prenatal exposure of rats to valproic acid reproduces the cerebellar anomalies associated with autism[END_REF][START_REF] Schneider | Behavioral alterations in rats prenatally exposed to valproic acid: animal model of autism[END_REF], and secondarily in mice [START_REF] Chapman | Sodium valproate: effects on social behaviour and physical development in the mouse[END_REF][START_REF] Roux | Regional and sex-dependent alterations in Purkinje cell density in the valproate mouse model of autism[END_REF]. This model is based on VPA administration during the early phases of pregnancy in mice or rats and it is characterized by the early appearance of motor, sensory and social disturbances, as well as increased repetitive behaviors [START_REF] Chapman | Sodium valproate: effects on social behaviour and physical development in the mouse[END_REF][START_REF] Ingram | Prenatal exposure of rats to valproic acid reproduces the cerebellar anomalies associated with autism[END_REF][START_REF] Roux | Regional and sex-dependent alterations in Purkinje cell density in the valproate mouse model of autism[END_REF][START_REF] Schneider | Behavioral alterations in rats prenatally exposed to valproic acid: animal model of autism[END_REF]. Similarly, the maternal immune activation model (MIA) induces motor, social and communication ASD-like deficits by injecting the viral mimic poly(I:C) [START_REF] Malkova | Neuroscience and Biobehavioral Reviews xxx (xxxx) 1-14 activation yields offspring displaying mouse versions of the three core symptoms of autism[END_REF] or the bacterial Lipopolysaccharide (LPS) [START_REF] Fernandez De Cossio | Prenatal infection leads to ASD-like behavior and altered synaptic pruning in the mouse offspring[END_REF] during pregnancy. The environmental approach has the advantage of having a sort of "construct validity", but has the main drawback of overlooking the major role of genetic factors in ASD etiopathology.

Indeed, another approach consists in manipulating the mouse genome to import into the animal model some of the genetic alterations associated with ASD, e.g., gene deletion/ mutations. The validity of this experimental strategy also has some problems, i.e., the heterogeneity of the genetic alterations known to be somehow involved in ASD, including basically almost all genes affecting neuronal function and development [START_REF] Abrahams | Advances in autism genetics: on the threshold of a new neurobiology[END_REF]. These genes code for molecules involved in: i) chromatin remodelling and regulation of transcription (e.g., MECP2, FMR1), ii) actin cytoskeleton dynamics (TSC1, TSC2, NF1) iii) synaptic scaffolding proteins (e.g., SHANK3), iv) receptors and transporters (e.g., GRIN2A, GRIK2, GABAR, SLC6A4, SLC25A13, OXTR, AVPR1), v) second-messenger systems (PRKCB1, CACNA1C, NBEA), vi) cell adhesion (e.g.,neuroligin NLGN3, NLGN4), and vii) secreted proteins (e.g., RELN, LAMB1) [reviewed in [START_REF] Persico | Searching for ways out of the autism maze: genetic, epigenetic and environmental clues[END_REF]]. Such list serves only to provide an indicative overview, as the number of implicated genes is supposed to be many-fold greater [START_REF] Buxbaum | Optimizing the phenotyping of rodent ASD models: enrichment analysis of mouse and human neurobiological phenotypes associated with high-risk autism genes identifies morphological, electrophysiological, neurological, and behavioral features[END_REF].

The role of all these genes in brain development is at the basis of a major hypothesis on autistic aetiology, i.e., the so-called "many genes, common pathways" [START_REF] Geschwind | Autism: many genes, common pathways?[END_REF], emphasizing the role of defective synaptic functioning and abnormal brain connectivity in ASD, both leading to altered information processing. One somewhat paradoxical finding about ASD is that, although there is proof that its aetiopathogenesis is the result of concomitant mutations in multiple loci, there is a growing list of genes whose disruption alone is sufficient for the whole autistic phenotype to occur. Based on such evidence, mouse models of single gene mutations may therefore be informative concerning the underlying mechanism(s) leading to ASD [START_REF] Ey | Behavioral profiles of mouse models for autism spectrum disorders[END_REF].

The last approach used to design animal models of ASD indeed focused on other developmental pathologies featuring ASD-like traits, but with a defined, single genetic cause and therefore well-established mouse models. A widely used example of this "simplified" approach is provided by the Fmr1-KO mouse line (The Dutch-Belgian Fragile X Consortium, 1994), modelling the most common monogenic cause of ASD, that is Fragile X syndrome (FXS), a X-linked disorder due to the lack of FMRP protein coded by the FMR1 gene [START_REF] Pieretti | Absence of expression of the FMR-1 gene in fragile X syndrome[END_REF]. Fmr1-KO mice display ASD-like deficits, i.e., reduced social interaction and social memory, deficits in reversal learning and ultrasonic communication alterations, as reviewed in [START_REF] Pietropaolo | Mouse models of fragile X syndrome[END_REF]. An other example of this approach focuses on Rett syndrome that is a monogenic disorder caused by deficiency of methyl-CpG-binding protein-2 (Mecp2) and characterized by ASD-like symptoms. Mice carrying null mutation for Mecp2 [START_REF] Chen | Deficiency of methyl-CpG binding protein-2 in CNS neurons results in a Rett-like phenotype in mice[END_REF], as well as various polymorphisms found in Rett patients [START_REF] Shahbazian | Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3[END_REF], display ASDlike phenotypes, including altered emission of ultrasonic vocalizations at infancy [START_REF] Picker | An altered neonatal behavioral phenotype in Mecp2 mutant mice[END_REF], and deficits in social and cognitive function in adulthood [START_REF] Moretti | Abnormalities of social interactions and home-cage behavior in a mouse model of Rett syndrome[END_REF]. A criticism to this "monogenic" research strategy comes from the consideration that the genetic mutations induced in these models are not invariably present in ASD patients and may therefore be valid in modelling only a specific subset of patients [START_REF] Budimirovic | What can we learn about autism from studying fragile X syndrome?[END_REF]. Nonetheless, recent research on pre-clinical models increasingly supports the view that investigating the consequences of single gene disruption may constitute a valuable tool for understanding ASD [START_REF] Wohr | Ultrasonic vocalizations in Shank mouse models for autism spectrum disorders: detailed spectrographic analyses and developmental profiles[END_REF][START_REF] Wohr | Behavioural methods used in rodent models of autism spectrum disorders: current standards and new developments[END_REF], a research approach that we have also followed in a major part of our research work based on the Fmr1-KO mouse line [START_REF] Bernardet | Fmr1 KO mice as a possible model of autistic features[END_REF]Oddi et al., 2013a), together with others' studies [START_REF] Budimirovic | What can we learn about autism from studying fragile X syndrome?[END_REF][START_REF] Hulbert | Monogenic mouse models of autism spectrum disorders: common mechanisms and missing links[END_REF][START_REF] Verma | Understanding intellectual disability and autism spectrum disorders from common mouse models: synapses to behaviour[END_REF].

In this complex and somehow undefined field of research, basically all these approaches to model ASD in laboratory mice have been employed to try to identify potential therapeutic targets to treat the disorder. Here, we will take advantage of examples of these different strategies to design mouse models of ASD to summarize the experimental evidence supporting the role of the endocannabinoid system (ECS) in the etiopathology of ASD. To this end, we will first review studies showing abnormalities in the functionality of the ECS in rodent models of ASD (Table 1), including several examples from the different approaches described above, e.g., based on strain differences (the BTBR mouse), prenatal exposure to environmental agents (the VPA or LPS models), candidate genes (NLGN-3 mutant mice), or monogenic developmental pathologies (the Fmr1-KO mouse). We will also summarize available data showing the correction of ASD-like behaviors in these models following pharmacological modulation of the ECS (Table 2), thus suggesting potential novel pharmacological approaches to ASD. Finally, we will discuss the possibility of inducing ASD-like behaviors in mice through pharmacological or genetic manipulations of the ECS (Table 3), thus discussing potential novel models for ASD. In line with the most prominent part of our experimental work on ECS and ASD, we will try as much as possible to keep our focus on the mouse species, though rat studies will be also included when necessary (e.g., for VPA and other environmental models). Our conclusive remarks will underline the relevance of extensive and refined behavioral analysis in assessing the role of the ECS in ASD.

Why should the endocannabinoid system play a role in ASD?

A converging etiopathological hypothesis for ASD: characteristics of the endocannabinoid system (ECS)

Recent studies have suggested that the multitude of possible causal factors involved in the pathogenic mechanisms of ASD may functionally converge into a relatively small subset of functional pathways that may represent an easier and more promising target for basic research on the disorder [START_REF] Zoghbi | Synaptic dysfunction in neurodevelopmental disorders associated with autism and intellectual disabilities[END_REF]. The functional convergence on particular signaling pathways and the shared synaptopathology of ASD raise the hope that similar therapeutic strategies may be effective for different forms of ASD which are related, but genetically distinct [START_REF] Zoghbi | Synaptic dysfunction in neurodevelopmental disorders associated with autism and intellectual disabilities[END_REF]. This "reductionist" approach is in line with the hypothesis of "many genes, common pathways" mentioned above [START_REF] Geschwind | Autism: many genes, common pathways?[END_REF]. In this context of searching for common causal pathways for ASD, the endocannabinoid system (ECS) seems a promising candidate to play a role in the etiopathology of this disorder and to provide a novel target of therapeutic interest. The ECS is an important modulator of neuronal functions, as demonstrated by the abundance of the cannabinoid CB1 receptors (CB1r) in the brain [START_REF] Mackie | Distribution of cannabinoid receptors in the central and peripheral nervous system[END_REF]. These are particularly enriched in cortex, hippocampus, amygdala, and cerebellum, i.e., brain areas that are strongly involved in the development of ASD. Furthermore, endocannabinoid signaling also regulates synaptogenesis and neuronal interconnectivity during development [START_REF] Berghuis | Hardwiring the brain: endocannabinoids shape neuronal connectivity[END_REF], processes whose defects are considered to be key mechanisms underlying ASD pathology [START_REF] Pardo | The neurobiology of autism[END_REF].

CB1 receptors are located in different cell types in the brain, including neurons and glial cells (Busquets- [START_REF] Busquets-Garcia | CB1 receptor signaling in the brain: extracting specificity from ubiquity[END_REF]. As illustrated Pietropaolo and G. Marsicano Neuroscience and Biobehavioral Reviews xxx (xxxx) 1-14 in Fig. 1, CB1r are mainly expressed pre-synaptically in both GABAergic and glutamatergic neurons where they are activated by their endogenous ligands, i.e., the endocannabinoids (ECs): anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG). These endogenous cannabinoids are produced at post-synaptic site by membrane depolarization or activation of metabotropic receptors [START_REF] Piomelli | More surprises lying ahead. The endocannabinoids keep us guessing[END_REF][START_REF] Piomelli | Endogenous cannabinoid signaling[END_REF]. Indeed calcium elevation generated by these two type of events is able to trigger plasma membrane lipid remodeling which generates as final products AEA, by the biosynthetic enzyme N-acyl phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), and 2-AG, by the Diacylglycerol lipase alpha (DAGL-a) [START_REF] Piomelli | The molecular logic of endocannabinoid signalling[END_REF]. The retrograde binding of AEA and 2-AG to pre-synaptic CB1r can trigger several intracellular pathways to reduce intracellular calcium and inhibit neurotransmitter release [START_REF] Piomelli | The molecular logic of endocannabinoid signalling[END_REF]. Endocannabinoids are terminated by re-uptake mechanisms; AEA is degraded by fatty acid amide hydrolase (FAAH), and 2-AG by monoacyl-glycerol lipase (MAGL). The ECS differs from classical neurotransmitter systems for the fact that there is no constitutive tone, but on-demand production of ECs, CB1r activation followed by immediate ECs degradation [START_REF] Piomelli | The molecular logic of endocannabinoid signalling[END_REF].

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Modulating the functionality of the ECS

The ECS is therefore a neuromodulatory network of lipid signaling pathways, including the endocannabinoids together with their recep-tors and the associated metabolic enzymes [START_REF] Di Marzo | The endocannabinoid system and its modulation by phytocannabinoids[END_REF]. Although endocannabinoids exert their central effects mainly through the cannabinoid type 1 receptor CB1r, they also interact with the other G-protein coupled cannabinoid type 2 receptor (CB2r), mostly involved in inflammatory processes and in the modulation of the immune system [START_REF] Jordan | Progress in brain cannabinoid CB2 receptor research: from genes to behavior[END_REF][START_REF] Turcotte | The CB2 receptor and its role as a regulator of inflammation[END_REF]. Additionally, they can also interact with non-CB1r/CB2r targets, e.g., the transient receptor potential vanilloid type 1 (TRPV1) channel, peroxisome proliferator-activated receptor (PPAR)-alpha, and PPAR-gamma as well as the G protein-coupled receptor GPR55 [START_REF] Pistis | From surface to nuclear receptors: the endocannabinoid family extends its assets[END_REF].

Several pharmacological tools are available for studying the ECS (mostly mentioned in Tables 2 and3), as extensively described elsewhere [START_REF] An | Targeting cannabinoid receptors: current status and prospects of natural products[END_REF]. Most commonly, the ECS is activated by CBr agonists, e.g., the natural cannabinoid Δ(9)-tetrahydrocannabinol (THC) and the synthetic WIN 55,212-2, ACEA, or CP-55,940 [START_REF] Griffin | Evaluation of cannabinoid receptor agonists and antagonists using the guanosine-5'-O-(3-[35S] thio)-triphosphate binding assay in rat cerebellar membranes[END_REF], or indirectly by pharmacological inhibition of AEA and 2-AG reuptake and degradation (e.g., URB597, . The latter approaches are useful for activity-dependent potentiation of endocannabinoid signals and would not have an effect in the absence of endocannabinoids production as normally happens with systemic CBr agonists administration [START_REF] Monory | Genetic models of the endocannabinoid system[END_REF]. The ECS can be blocked mainly by CBr antagonists (e.g., rimonabant or SR141716A, AM251) or inhibition of endocannabinoids biosynthesis [e.g., DO-34 for DAGL [START_REF] Mitra | A role for the endocannabinoid enzymes monoacylglycerol and diacylglycerol lipases in cue-induced cocaine craving following prolonged abstinence[END_REF] and LEI-401 for AEA [START_REF] Mock | Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice[END_REF]]. In addition, transgenic mouse models with constitutive and cell type specific [START_REF] Haller | CB1 cannabinoid receptors mediate anxiolytic effects: convergent genetic and pharmacological evidence with CB1specific agents[END_REF][START_REF] Haring | Cannabinoid type-1 receptor signaling in central serotonergic neurons regulates anxiety-like behavior and sociability[END_REF][START_REF] Haring | Circuit specific functions of cannabinoid CB1 receptor in the balance of investigatory drive and exploration[END_REF][START_REF] Fyke | Communication and social interaction in the cannabinoid-type 1 receptor null mouse: implications for autism spectrum disorder[END_REF][START_REF] Gould | Acetaminophen differentially enhances social behavior and cortical cannabinoid levels in inbred mice[END_REF][START_REF] Terzian | Cannabinoid receptor type 1 receptors on GABAergic vs. glutamatergic neurons differentially gate sex-dependent social interest in mice[END_REF]Uriguen et al., 2004) 3). These genetically modified engineered mice have allowed identifying a plethora of ECSdependent regulation of brain and peripheral functions [START_REF] Cristino | Cannabinoids and the expanded endocannabinoid system in neurological disorders[END_REF], involving different cell types and cellular processes (Busquets- [START_REF] Busquets-Garcia | Dissecting the cannabinergic control of behavior: the where matters[END_REF]. Pietropaolo and G. Marsicano Neuroscience and Biobehavioral Reviews xxx (xxxx) 1-14

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3. Clinical and pre-clinical evidence of ECS alterations in ASD: identifying novel therapeutic strategies

Alterations in ECS functionality in ASD patients and related pharmacological approaches

An etiopathological role of alterations in ECS functionality has been already suggested for several psychiatric [START_REF] Rubino | Endocannabinoids and mental disorders[END_REF] and neurological disorders [START_REF] Iannotti | Endocannabinoids and endocannabinoidrelated mediators: targets, metabolism and role in neurological disorders[END_REF], such as schizophrenia, anxiety, depression, neurodegenerative diseases, epilepsy and others [START_REF] Cristino | Cannabinoids and the expanded endocannabinoid system in neurological disorders[END_REF]. This is not surprising, considering the major impact of the ECS in the modulation of neuronal and synaptic functions, as well as of brain development. Indeed, a clear contribution of the ECS has been demonstrated on behavioral functions that are altered in several neuropathologies, including ASD. The impairments span across cognitive and emotional abilities, circadian rhythms, epileptic seizures and social interaction [e.g., [START_REF] Marco | The endocannabinoid system in the regulation of emotions throughout lifespan: a discussion on therapeutic perspectives[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF][START_REF] Trezza | Endocannabinoids in amygdala and nucleus accumbens mediate social play reward in adolescent rats[END_REF][START_REF] Vaughn | Endocannabinoid signalling: has it got rhythm?[END_REF]]. Beside this indirect evidence, recent findings have provided a more direct link between ECS functionality and ASD. Human neuroimaging studies have for instance demonstrated that variations of the cannabinoid receptor gene are involved in the modulation of social reward responsivity, specifically in the striatal response to happy faces [START_REF] Chakrabarti | Variations in the human cannabinoid receptor (CNR1) gene modulate striatal responses to happy faces[END_REF], a function which is specifically altered in ASD patients.

As illustrated in Fig. 1, several alterations in ECS functionality have been described in ASD patients. Post-mortem analyses showed reduced expression of CB1r in several areas of ASD brains [START_REF] Purcell | Postmortem brain abnormalities of the glutamate neurotransmitter system in autism[END_REF], while recent studies reported that AEA plasmatic concentrations were significantly lower in ASD children compared to controls [START_REF] Karhson | Plasma anandamide concentrations are lower in children with autism spectrum disorder[END_REF], in the absence of changes in blood levels of 2-AG (Aran et al., 2019b). It is still not clear whether the reduced AEA levels could be due to a reduced activity of NAPE-PLD, the enzyme that synthesizes AEA [START_REF] Schultz | Endocannabinoid system dysregulation from acetaminophen use may lead to autism spectrum disorder: could cannabinoid treatment be efficacious?[END_REF], or increased activity of FAAH, the enzyme that degrades them. Nonetheless, it is increasingly evident that AEA circulating levels can represent a useful biomarker for the early identification of subpopulations of affected patients and individual at risk and to design specific therapeutic strategies. Circulating ECs could indeed contribute to predict autism risk prior to the onset of behavioral abnormalities and to initiate early interventions, since alterations in AEA levels appeared to be inversely correlated with age in ASD patients, as the most marked reductions were detected in ASD children (Aran et al., 2019b;[START_REF] Karhson | Plasma anandamide concentrations are lower in children with autism spectrum disorder[END_REF]. Also, it has been suggested that a decrease in the levels of circulating endocannabinoids may be a central mechanism linking certain environmental risk factors to ASD, e.g., early exposure to pharmacological agents [START_REF] Schultz | Endocannabinoid system dysregulation from acetaminophen use may lead to autism spectrum disorder: could cannabinoid treatment be efficacious?[END_REF]. Treatments compensating the reduced EC tone would then be a possible valuable strategy to rescue or prevent these environmentally-induced forms of ASD [START_REF] Schultz | Endocannabinoid system dysregulation from acetaminophen use may lead to autism spectrum disorder: could cannabinoid treatment be efficacious?[END_REF].

Taking into account the relevance of the endocannabinoids in ASD, it is not surprising that some phytocannabinoids, such as Cannabidiol (CBD), have been suggested as therapeutic strategies for this disorder [START_REF] Poleg | Cannabidiol as a suggested candidate for treatment of autism spectrum disorder[END_REF]. It has been indeed hypothesized that its therapeutic effects could be due to the inhibition of FAAH [START_REF] Di Marzo | The endocannabinoid system and its modulation by phytocannabinoids[END_REF] and therefore a compensation of the low levels of AEA found in ASD patients (Aran et al., 2019b;[START_REF] Schultz | Endocannabinoid system dysregulation from acetaminophen use may lead to autism spectrum disorder: could cannabinoid treatment be efficacious?[END_REF]. Once again, the assessment of EC levels could be a useful tool to screen sub-populations of ASD patients who would more markedly benefit from treatments with phytocannabinoids, as suggested by some authors [START_REF] Schultz | Endocannabinoid system dysregulation from acetaminophen use may lead to autism spectrum disorder: could cannabinoid treatment be efficacious?[END_REF].

Several clinical studies demonstrated the therapeutic effects of CBD in pathological conditions associated with ASD, including social phobia [START_REF] Bergamaschi | Cannabidiol reduces the anxiety induced by simulated public speaking in treatment-naive social phobia patients[END_REF] and epilepsy [START_REF] Devinsky | Cannabidiol in patients with treatment-resistant epilepsy: an open-label interventional trial[END_REF], as well as for the ASD and cognitive symptoms of Fragile X patients [START_REF] Heussler | A phase 1/2, open-label assessment of the safety, tolerability, and efficacy of transdermal cannabidiol (ZYN002) for the treatment of pediatric fragile X syndrome[END_REF][START_REF] Tartaglia | Treatment of fragile X syndrome with cannabidiol: a case series study and brief review of the literature[END_REF]. Moreover, there are preliminary reports of beneficial effects of CBD in idiopathic ASD itself [START_REF] Barchel | Oral cannabidiol use in children with autism spectrum disorder to treat related symptoms and co-morbidities[END_REF]. For instance, recent clinical studies demonstrated that CBD-rich cannabis (with a ratio of CBD:THC of 20:1) reduced behavioral outbreaks in children with ASD and severe behavioral problems (Aran et al., 2019a), and significantly reduced disruptive behavior and secondary autistic measures in ASD patients [START_REF] Aran | Cannabinoid treatment for autism: a proof-ofconcept randomized trial[END_REF], such as alterations in Social Responsiveness Scale and Autism Parenting Stress Index (in the absence of treatment-related serious adverse events). Furthermore, a study using resting state fMRI demonstrated that the brain response to a single oral administration of CBD differed between individuals with and without ASD. CBD significantly altered the functional connectivity of the cerebellar vermis with several of its subcortical (striatal) and cortical targets [START_REF] Pretzsch | The effect of cannabidiol (CBD) on low-frequency activity and functional connectivity in the brain of adults with and without autism spectrum disorder (ASD)[END_REF], but only in ASD patients, suggesting the efficacy of this phytocannabinoid in treating the abnormal levels of cerebellar excitation-inhibition levels, known to be correlated with abnormal cerebro-cerebellar functional connectivity in patients with ASD [START_REF] Hegarty | Cerebro-cerebellar functional connectivity is associated with cerebellar excitation-inhibition balance in autism spectrum disorder[END_REF].

Furthermore, rare genetic variants in the core endocannabinoid system genes (e.g., coding for CB1r, CB2r, DAGL-α, FAAH and MAGL) were identified in molecular testing data from a large sample of patients with a broad spectrum of neurological disorders, showing that heterozygous rare variants in DAGL-α were significantly associated with seizures and ASD [START_REF] Smith | Rare genetic variants in the endocannabinoid system genes CNR1 and DAGLA are associated with neurological phenotypes in humans[END_REF]. In addition, increased expression of CB2 receptors has been described in blood monocytes of ASD patients [START_REF] Siniscalco | Cannabinoid receptor type 2, but not type 1, is up-regulated in peripheral blood mononuclear cells of children affected by autistic disorders[END_REF], suggesting a role of the ECS in mediating the immune alterations of ASD pathology. CB2r is indeed able to modulate development, migration, proliferation, and effector functions of immune cells [START_REF] Basu | Unraveling the complexities of cannabinoid receptor 2 (CB2) immune regulation in health and disease[END_REF], and alterations in immune system have been reported in ASD patients [START_REF] Ormstad | Role of the immune system in autism spectrum disorders (ASD)[END_REF][START_REF] Siniscalco | Inflammation and neuroimmune dysregulations in autism spectrum disorders[END_REF]. Overall, the existing clinical data on ECS alterations point towards a reduced endocannabinoid tone which might then be the leading cause for the neuronal hyper-excitability often associated to ASD (Fig. 1) [START_REF] Bozzi | Neurobiological bases of autism-epilepsy comorbidity: a focus on excitation/inhibition imbalance[END_REF][START_REF] Sohal | Excitation-inhibition balance as a framework for investigating mechanisms in neuropsychiatric disorders[END_REF]. Based on this large body of evidence from ASD patients, several studies have employed animal models in the last years to better evaluate the role of the ECS in the etiopathology of ASD.

Alterations of ECS in ASD animal models

Alterations in ECS functionality have been described in several animal models of ASD [reviewed also in [START_REF] Zamberletti | Cannabidivarin completely rescues cognitive deficits and delays neurological and motor defects in male Mecp2 mutant mice[END_REF]], based on the approaches described in Section 1.4 of the present review (Table 1).

The ASD model that has been the most extensively investigated in relation to the ECS is the Fmr1-KO mouse, since this is also a model of another major (but monogenic) developmental disorder associated with ASD, that is Fragile X syndrome (reviewed in [START_REF] Pietropaolo | Mouse models of fragile X syndrome[END_REF]. As Fragile X syndrome is also a developmental pathology lacking therapeutic targets, a large amount of research has been devoted on the role of the ECS in Fragile X syndrome on its own. Although the validity of the Fmr1-KO mouse to model ASD is still debatable, because the construct validity of this animal model is limited to Fragile X syndrome (i.e., mutation in the X-linked Fmr1 gene and lack of FMRP protein), its relevance to study ASD is nowadays widely recognized, since Fragile X syndrome represents the most common monogenic cause of ASD [START_REF] Chaste | High-functioning autism spectrum disorder and fragile X syndrome: report of two affected sisters[END_REF][START_REF] Clifford | Autism spectrum phenotype in males and females with fragile X full mutation and premutation[END_REF][START_REF] Harris | Autism profiles of males with fragile X syndrome[END_REF][START_REF] Kielinen | Associated medical disorders and disabilities in children with autistic disorder: a population-based study[END_REF][START_REF] Losh | Social communication and theory of mind in boys with autism and fragile x syndrome[END_REF][START_REF] Reddy | Cytogenetic abnormalities and fragile-X syndrome in Autism Spectrum Disorder[END_REF][START_REF] Toriello | Approach to the genetic evaluation of the child with autism[END_REF]. Nonetheless, caution should be taken in the strict application of the findings from the Fmr1-KO model to ASD, since it is possible that they better suit a distinct clinical subpopulation of ASD patients with Fragile X syndrome. Alterations in the ECS functionality have been demonstrated in Fmr1-KO mice by several studies, highlighting a complex imbalance in EC signaling. It was demonstrated that the coupling between mGluR activation and ECs mobilization was altered in Fmr1-KO mice in several brain areas [START_REF] Jung | Uncoupling of the endocannabinoid signalling complex in a mouse model of fragile X syndrome[END_REF][START_REF] Maccarrone | Abnormal mGlu 5 receptor/endocannabinoid coupling in mice lacking FMRP and BC1 RNA[END_REF][START_REF] Zhang | Enhanced endocannabinoid signaling elevates neuronal excitability in fragile X syndrome[END_REF], in the absence of alterations in the expression of Pietropaolo and G. Marsicano Neuroscience and Biobehavioral Reviews xxx (xxxx) 1-14 cannabinoid CB1r [START_REF] Zhang | Enhanced endocannabinoid signaling elevates neuronal excitability in fragile X syndrome[END_REF] and baseline levels of 2-AG [START_REF] Jung | Uncoupling of the endocannabinoid signalling complex in a mouse model of fragile X syndrome[END_REF]. Enhanced activity of diacylglycerol lipase (DAGL) was also reported in the dorsal striatum of these mutants [START_REF] Maccarrone | Abnormal mGlu 5 receptor/endocannabinoid coupling in mice lacking FMRP and BC1 RNA[END_REF]. This might provide a probable substrate for the enhanced glutamatergic activity often observed in the Fmr1-KO mouse model. We also obtained results supporting altered ECS functionality in Fmr1-KO mice, as demonstrated by the analysis of the typical behavioral effects of acute pharmacological challenge of the ECS in domains unrelated to ASD, using either the CB1r antagonist rimonabant, or an agonist, WIN 55,212-2 [START_REF] Pietropaolo | The Endocannabinoid System as a Therapeutic Target for Autism Spectrum disorders: Lessons from Behavioural Studies on Genetic Mouse Models, Miniconference "Behavioural Neuroscience for the Next Decade: why Behaviour Matters to Brain Science?[END_REF]. On one hand, Fmr1-KOs showed a similar reduction to WT littermates in fasting-induced food intake, i.e., a classical behavioral effect of acute rimonabant administration (3 mg/Kg, intraperitoneal) which has been shown to depend on peripheral CB1r [START_REF] Bellocchio | Activation of the sympathetic nervous system mediates hypophagic and anxiety-like effects of CB(1) receptor blockade[END_REF][START_REF] Gomez | A peripheral mechanism for CB1 cannabinoid receptor-dependent modulation of feeding[END_REF]. On the other hand, mutant mice showed a marked reduced classical behavioral (motor and sensory) response to the acute injection of WIN 55,212-2 [START_REF] Pietropaolo | The Endocannabinoid System as a Therapeutic Target for Autism Spectrum disorders: Lessons from Behavioural Studies on Genetic Mouse Models, Miniconference "Behavioural Neuroscience for the Next Decade: why Behaviour Matters to Brain Science?[END_REF], as assessed by the tetrad experiment (i.e., assessing the hypolocomotion, hypothermia, analgesia and catalepsy typically induced by a 3 mg/Kg dose of this CB1r agonist). These results suggest a hypo-responsiveness of central, but not peripheral CB1r in Fmr1-KO mice, that may explain the reduced activation by the exogenous agonists.

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A similar result was obtained in the BTBR mouse model for ASD, i.e., showing a reduced sensitivity to the sedative effects of high doses of CBr agonists, in this case, THC [START_REF] Onaivi | Consequences of cannabinoid and monoaminergic system disruption in a mouse model of autism spectrum disorders[END_REF]. The same authors showed upregulation of gene expression of CB2r in the cerebellum of untreated BTBR mice [START_REF] Liu | Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands[END_REF], an effect usually observed in C57BL6/mice following acute administration of sedative doses of WIN 55,212-2 [START_REF] Liu | Species differences in cannabinoid receptor 2 (CNR2 gene): identification of novel human and rodent CB2 isoforms, differential tissue expression and regulation by cannabinoid receptor ligands[END_REF]. BTBR mice also showed an enhancement of CB1r functionality in cortical [START_REF] Gould | Acetaminophen differentially enhances social behavior and cortical cannabinoid levels in inbred mice[END_REF] and hippocampal areas [START_REF] Gould | Enhanced novelty-induced corticosterone spike and upregulated serotonin 5-HT1A and cannabinoid CB1 receptors in adolescent BTBR mice[END_REF] without any reported impact on physiological ECS-mediated processes. Some authors suggested that CB1r upregulation could be related to the hyper-responsiveness of the HPA axis characterizing these mice [START_REF] Gould | Enhanced novelty-induced corticosterone spike and upregulated serotonin 5-HT1A and cannabinoid CB1 receptors in adolescent BTBR mice[END_REF]. The role of oxytocin was instead underlined by others [START_REF] Wei | Enhancement of anandamide-mediated endocannabinoid signaling corrects autism-related social impairment[END_REF], as this neuropeptide drives anandamide mobilization in some brain areas in the mouse, e.g., the nucleus accumbens (NAc), a brain structure that regulates motivated behavior [START_REF] Wei | Endocannabinoid signaling mediates oxytocin-driven social reward[END_REF]. Interestingly, AEA mobilization in this brain area is potentiated by social contact and inhibited by social isolation in mice [START_REF] Wei | Endocannabinoid signaling mediates oxytocin-driven social reward[END_REF].

Alterations in ECS functionality were also detected in mouse models based on the "candidate gene" approach. This is the case for instance of mouse models based on mutations in the cell-adhesion molecule neuroligin NLG3 that are implicated in monogenic heritable ASD. These mutant mice displayed at adulthood reduced ultrasound vocalization and a lack of social novelty preference, with variable cognitive deficits [START_REF] Radyushkin | Neuroligin-3-deficient mice: model of a monogenic heritable form of autism with an olfactory deficit[END_REF], together with increased occurrence of repetitive behaviors [START_REF] Rothwell | Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors[END_REF]. NLG3 mutants showed reduced CB1r-dependent modulation of GABAergic transmission in several brain areas, including the hippocampus [START_REF] Foldy | Autism-associated neuroligin-3 mutations commonly disrupt tonic endocannabinoid signaling[END_REF], cortex [START_REF] Speed | Increased cortical inhibition in autism-linked neuroligin-3R451C mice is due in part to loss of endocannabinoid signaling[END_REF], dorsal striatum [START_REF] Martella | The neurobiological bases of autism spectrum disorders: the R451C-neuroligin 3 mutation hampers the expression of long-term synaptic depression in the dorsal striatum[END_REF] and amygdala [START_REF] Hosie | Altered amygdala excitation and CB1 receptor modulation of aggressive behavior in the neuroligin-3(R451C) mouse model of autism[END_REF], but not in the nucleus accumbens [START_REF] Rothwell | Autism-associated neuroligin-3 mutations commonly impair striatal circuits to boost repetitive behaviors[END_REF]. These findings suggest the presence of region-specific alterations in the balance of excitatory and inhibitory synaptic transmission (E/I balance), a mechanism known to play a central role in the etiopathology of ASD.

Finally, evidence for the involvement of the ECS in ASD was obtained from environmental-based models, i.e. the VPA and LPS models, although to our knowledge all studies on this issue were conducted in rats. Rats prenatally exposed to VPA displayed altered expression of phosphorylated CB1r in the amygdala, hippocampus and dorsal striatum [START_REF] Kerr | Alterations in the endocannabinoid system in the rat valproic acid model of autism[END_REF]. Since alterations in CB1r phosphorylation may reflect abnormalities in CB1r-activation [START_REF] Daigle | Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism[END_REF], it has been suggested that the changes observed in VPA rats may reveal a compensatory response aimed at normalizing CB1-mediated signaling and the relative imbalance of the EC system [START_REF] Garcia | Protein kinase C disrupts cannabinoid actions by phosphorylation of the CB1 cannabinoid receptor[END_REF], as suggested by the reduced expression of DAGL-α, and NAPE-PLD and FAAH in VPA brains [START_REF] Kerr | Alterations in the endocannabinoid system in the rat valproic acid model of autism[END_REF][START_REF] Servadio | Targeting anandamide metabolism rescues core and associated autistic-like symptoms in rats prenatally exposed to valproic acid[END_REF]. In LPStreated rats, alterations similar to those observed in ASD patients were found in the amygdala, including reduced CB1r expression and increased FAAH levels [START_REF] Doenni | Deficient adolescent social behavior following early-life inflammation is ameliorated by augmentation of anandamide signaling[END_REF].

Pharmacological targeting of the ECS in ASD models

The data from mouse models of ASD support the presence of alterations in the functionality of the ECS, involving multiple components of the systems, sometimes with different modalities according to the considered model. Nonetheless, a common aspect of multiple animal models relies in an imbalance in EC signaling, suggesting the therapeutic relevance of its correction by targeting different components of the ECS, including the functionality of CB receptors as well as the synthesis/degradation of the 2-AG and AEA (as illustrated in Table 2).

When the impact of pharmacological manipulations of CB receptors was studied, the effects seem to critically depend on the type of animal models and of the behavioral alterations analyzed. In Fmr1-KO mice, CB1Rr blockade (also through genetic approaches, i.e., generation of double knock-outs for CB1r and Fmr1) eliminated the cognitive and nociceptive alterations together with the enhanced susceptibility to audiogenic seizures, while the pharmacological blockade of CB2R rescued anxiety abnormalities (Busquets- [START_REF] Busquets-Garcia | Targeting the endocannabinoid system in the treatment of fragile X syndrome[END_REF]Garcia et al., , 2014)). As some of these beneficial effects were accompanied by normalization of overactivated mTOR signalling and were observed also following pharmacological inhibition of mTOR or mGluR5 in the same mutant mice, some authors have suggested that mTOR pathway -regulating cell growth, proliferation/survival, and metabolism [START_REF] Lipton | The neurology of mTOR[END_REF][START_REF] Wipperman | Mammalian target of rapamycin: a metabolic rheostat for regulating adipose tissue function and cardiovascular health[END_REF]-may represent a key mechanism to explain the role of ECS in ASD (Busquets- Garcia et al., 2014). The same authors have supported the potential therapeutic applications of rimonabant for FXS and ASD, although beneficial effects were described only on the cognitive ASD-relevant deficits of Fmr1-KO mice [START_REF] Gomis-Gonzalez | Possible therapeutic doses of cannabinoid type 1 receptor antagonist reverses key alterations in fragile X syndrome mouse model[END_REF]. Given that the same authors failed to detect alterations in LTD in the hippocampus of Fmr1-KO mice (Busquets- [START_REF] Busquets-Garcia | Targeting the endocannabinoid system in the treatment of fragile X syndrome[END_REF]Garcia et al., , 2014)), it is possible that ECS blockade might impact on FMR1-KO cognitive deficits not by acting on CB1-dependent synaptic plasticity alterations, but merely on intracellular signaling mechanisms. This hypothesis may explain the discrepancies obtained from other ASD mouse models, e.g., the BTBR [START_REF] Wei | Enhancement of anandamide-mediated endocannabinoid signaling corrects autism-related social impairment[END_REF] and NLG-3 [START_REF] Speed | Increased cortical inhibition in autism-linked neuroligin-3R451C mice is due in part to loss of endocannabinoid signaling[END_REF] lines, where no behavioral rescue or region-specific brain effects were induced by CBr-inhibitors (Table 2).

The use of direct or indirect CBr agonists seems instead a more convincing pharmacological approach, in line with the evidence of an overall reduced functionality of the ECS in ASD, supported by clinical and pre-clinical studies (see Fig. 1 and Table 1). In NL-3 mice, the administration of CB1r synthetic agonists induced a partial rescue of striatal LTD [START_REF] Martella | The neurobiological bases of autism spectrum disorders: the R451C-neuroligin 3 mutation hampers the expression of long-term synaptic depression in the dorsal striatum[END_REF], as well as beneficial behavioral effects, i.e., a significant reduction in aggressive behavior [START_REF] Hosie | Altered amygdala excitation and CB1 receptor modulation of aggressive behavior in the neuroligin-3(R451C) mouse model of autism[END_REF]. Nonetheless, despite the efforts of considerably lowering the doses [START_REF] Gomis-Gonzalez | Possible therapeutic doses of cannabinoid type 1 receptor antagonist reverses key alterations in fragile X syndrome mouse model[END_REF], it is unlikely that therapeutic strategies directly targeting central CB receptors may still be actively investigated, due to the risk of severe adverse psychiatric effects, e.g., appearance of anxiety, depression and suicidal thoughts [START_REF] Akbas | A critical review of the cannabinoid receptor as a drug target for obesity management[END_REF]. Nevertheless, the neurosteroid pregnenolone was recently shown to be produced upon excessive CB1 receptor activation and to act as a signalspecific allosteric endogenous inhibitor of the receptor itself, thereby providing protection against ECs overload [START_REF] Vallee | Pregnenolone can protect the brain from cannabis intoxication[END_REF]. Thus, pregnenolone-derivatives exploiting this mechanism of action are currently being tested in clinical settings and might provide a much safer way to inhibit CB1 receptor activity than orthosteric synthetic antagonists.

U N C O R R E C T E D P R O O F

A stronger interest has been instead devoted to target the endocannabinoids, AEA in particular, since they emerge as a common altered element across multiple models (including ASD patients) and therefore a potential central factor in the etiopathology of ASD (Fig. 1). Administration of inhibitors of FAAH (e.g., URB597), thus increasing AEA levels, has shown to eliminates the cognitive [START_REF] Qin | Endocannabinoidmediated improvement on a test of aversive memory in a mouse model of fragile X syndrome[END_REF] and social deficits of Fmr1-KO mice [START_REF] Wei | Enhancement of anandamide-mediated endocannabinoid signaling corrects autism-related social impairment[END_REF]. Also, enhancing 2-AG signaling through administration of JZL184, a pharmacological inhibitor of the 2-AG degrading enzyme MAGL, corrected some of the Fmr1 mutants' behavioral alterations, e.g. hyperactivity and anxiety abnormalities [START_REF] Jung | Uncoupling of the endocannabinoid signalling complex in a mouse model of fragile X syndrome[END_REF], without effects on the cognitive alterations [START_REF] Qin | Endocannabinoidmediated improvement on a test of aversive memory in a mouse model of fragile X syndrome[END_REF], that once again seem to have an independent modulation in this model. The therapeutic value of FAAH inhibition was observed also in BTBR mice, where it rescued their social impairments. Interestingly, this effect was independent from changes in anxiety-levels and was prevented by CB1r blockade [START_REF] Wei | Enhancement of anandamide-mediated endocannabinoid signaling corrects autism-related social impairment[END_REF]. Enhancing AEA signaling through inhibition of its degradation was also effective in mitigating the behavioral phenotypes induced by prenatal VPA [START_REF] Kerr | Pharmacological inhibition of fatty acid amide hydrolase attenuates social behavioural deficits in male rats prenatally exposed to valproic acid[END_REF][START_REF] Servadio | Targeting anandamide metabolism rescues core and associated autistic-like symptoms in rats prenatally exposed to valproic acid[END_REF] or LPS [START_REF] Doenni | Deficient adolescent social behavior following early-life inflammation is ameliorated by augmentation of anandamide signaling[END_REF] exposure in environmental rat models of ASD.

In line with the relevance of the endocannabinoids as a therapeutic target in ASD, some phytocannabinoids, such as CBD have been suggested as treatments for this syndrome [START_REF] Poleg | Cannabidiol as a suggested candidate for treatment of autism spectrum disorder[END_REF], as previously mentioned. The high interest in CBD and its derived compounds, e.g., cannabidivarin (CBDV), stems from the fact that these molecules do not induce the noxious psychotropic effects typical of directly targeting central CB receptors, as for example THC [START_REF] Agarwal | Current state of evidence of cannabis utilization for treatment of autism spectrum disorders[END_REF]. Furthermore, CBDV has powerful anti-inflammatory properties [START_REF] Burstein | Cannabidiol (CBD) and its analogs: a review of their effects on inflammation[END_REF], probably due to its action as agonist of transient receptor potential channels [START_REF] De Petrocellis | Neuroscience and Biobehavioral Reviews xxx (xxxx) 1-14 extracts on TRP channels and endocannabinoid metabolic enzymes[END_REF]. Hence, several preclinical studies have been conducted to evaluate the therapeutic efficacy of CBDV in animal models of ASD. For instance, chronic systemic administration of CBDV rescued the social deficits as well as the brain alterations of MeCP2-308 mice [START_REF] Vigli | Chronic treatment with the phytocannabinoid Cannabidivarin (CBDV) rescues behavioural alterations and brain atrophy in a mouse model of Rett syndrome[END_REF], a validated model of Rett syndrome. CBDV also eliminated the cognitive deficits of Mecp2 mice, concomitantly normalizing the upregulation of brain CB1r and CB2r, thus further supporting the role of the ECS in this ASD-related developmental disorder (Zamberletti et al., 2019a).

CBDV also rescued the cognitive, social and repetitive alterations shown by VPA rats, restoring hippocampal endocannabinoid signaling and neuroinflammation induced by prenatal VPA exposure (Zamberletti et al., 2019b). Our unpublished results from Fmr1-KO mice also supported the efficacy of CBDV chronic treatments for ASDlike behavioral alterations, although the early timing of intervention appeared to be crucial [START_REF] Pietropaolo | The Endocannabinoid System as a Therapeutic Target for Autism Spectrum disorders: Lessons from Behavioural Studies on Genetic Mouse Models, Miniconference "Behavioural Neuroscience for the Next Decade: why Behaviour Matters to Brain Science?[END_REF]. The efficacy of CBDV was in fact markedly higher when the treatment was administered starting at weaning than at adulthood in our Fmr1-KO mice. Hence, at least some behavioral alterations may be irreversible by CBDV beyond certain age of development. It remains to be tested if the therapeutic window we highlighted, including the adolescent age (4-7 weeks of age in rodents), may be generalized to other modalities targeting the ECS. Adolescence is an early life phase that is indeed characterized by high levels of neurobehavioral plasticity in rodents as well as in humans [START_REF] Spear | The adolescent brain and age-related behavioral manifestations[END_REF], and is easier to target with pharmacological interventions compared to earlier developmental phases (where the mother-pup interaction has a major confounding impact).

ASD-like phenotypes induced by manipulations of the endocannabinoid system: designing novel animal models for ASD

Behavioral effects of pharmacological targeting the ECS on ASDrelevant domains in WT animals

The recent predominant research focus on genetic mouse models of ASD has somehow attenuated the interest in pharmacological ap-proaches, though they may provide complementary precious information on the etiopathology of this disorder. Evaluating the ASD-like behavioral effects of pharmacological manipulations of the ECS in wildtype rodents can in fact contribute to shed light on the involvement of this complex neuromodulatory system in this developmental disease. Cannabis intoxication has been shown in humans and laboratory animals to share several symptoms with ASD, such as cognitive and somatosensory impairment, social withdrawal and repetitive behavior [START_REF] Zehra | Cannabis addiction and the brain: a review[END_REF]. However little is still known on the effects of recreational, non-intoxicating doses of cannabinoids on ASD-relevant behaviors, i.e., social behavior. In humans, recreational doses of THC have been shown to modulate the perception of social threats in a recent functional magnetic resonance imaging study [START_REF] Phan | Cannabinoid modulation of amygdala reactivity to social signals of threat in humans[END_REF], by significantly reducing amygdala reactivity without affecting primary visual and motor cortical areas. A recent study has suggested an association between maternal cannabis use in pregnancy and the incidence of autism spectrum disorder in the offspring [START_REF] Corsi | Maternal cannabis use in pregnancy and child neurodevelopmental outcomes[END_REF].

To date, the evaluation of the behavioral effects of administration of CBr agonists, e.g., THC or synthetic cannabinoids suspensions yielded to contradictory results in wild-type mice [e.g., [START_REF] Almeida | Effects of cannabinoid and vanilloid drugs on positive and negative-like symptoms on an animal model of schizophrenia: the SHR strain[END_REF][START_REF] Argue | Activation of both CB1 and CB2 endocannabinoid receptors is critical for masculinization of the developing medial amygdala and juvenile social play behavior[END_REF][START_REF] Gould | Acetaminophen differentially enhances social behavior and cortical cannabinoid levels in inbred mice[END_REF][START_REF] Pietropaolo | Chronic cannabinoid receptor stimulation selectively prevents motor impairments in a mouse model of Huntington's disease[END_REF][START_REF] Tambaro | Long-term CB(1) receptor blockade enhances vulnerability to anxiogenic-like effects of cannabinoids[END_REF]], displaying either pro or antisocial effects, according to the dose and the type of tests employed (Table 3). One possible reason for this discrepancy might be the differential expression and activation of CB1r in different neuronal populations. Indeed an elegant series of studies shed light on the biphasic effects of cannabinoids on both feeding and anxiety [START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Rey | Biphasic effects of cannabinoids in anxiety responses: CB1 and GABA(B) receptors in the balance of GABAergic and glutamatergic neurotransmission[END_REF][START_REF] Ruehle | The endocannabinoid system in anxiety, fear memory and habituation[END_REF] showing that opposite effects of different doses were mediated by activation of CB1r on different neuronal populations, either glutamatergic or GABAergic [START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Ruehle | The endocannabinoid system in anxiety, fear memory and habituation[END_REF]. A similar scenario may apply also to the effects of cannabinoids on sociability, as suggested by recent studies on conditional knockout mice for CB1r in different neuronal types [START_REF] Haring | Cannabinoid type-1 receptor signaling in central serotonergic neurons regulates anxiety-like behavior and sociability[END_REF][START_REF] Haring | Circuit specific functions of cannabinoid CB1 receptor in the balance of investigatory drive and exploration[END_REF][START_REF] Terzian | Cannabinoid receptor type 1 receptors on GABAergic vs. glutamatergic neurons differentially gate sex-dependent social interest in mice[END_REF]. Recently, it has emerged a novel link between THC and social interest, involving CB1r in non-neuronal populations, i.e., in astrocytes [START_REF] Jimenez-Blasco | Glucose metabolism links astroglial mitochondria to cannabinoid effects[END_REF]. This innovative work has demonstrated that 24 h after THC administration, the activation of CB1r located in association with mitochondrial membranes in astrocytes induces a downregulation of glucose metabolism, thereby decreasing the amount of lactate that is shuttled to neurons. This in turn causes oxidative and bioenergetic stress in the neurons and induces the appearance of social withdrawal in mice [START_REF] Jimenez-Blasco | Glucose metabolism links astroglial mitochondria to cannabinoid effects[END_REF]. It will be interesting to investigate if similar mechanisms are involved in the pathophysiological mechanisms underlying social alterations typical of ASD.

An alternative pharmacological approach to activate the ECS rather than administration of CB1r agonists consists of promoting endogenous endocannabinoids' signalling (Table 3), for instance by enhancing AEA brain levels (as already mentioned above). The administration of AEA hydrolysis inhibitors (e.g.,URB597) either systemically or directly into brain limbic areas (e.g., amygdala and nucleus accumbens), has been reported for instance to increase social activity in rats [START_REF] Trezza | Endocannabinoids in amygdala and nucleus accumbens mediate social play reward in adolescent rats[END_REF] and reduced the anxiety induced by social defeat in mice [START_REF] Rossi | Preservation of striatal cannabinoid CB1 receptor function correlates with the antianxiety effects of fatty acid amide hydrolase inhibition[END_REF]. Prosocial effects were also detected following administration of JZL-84 in WT rats and mice [START_REF] Manduca | Interacting cannabinoid and opioid receptors in the nucleus accumbens core control adolescent social play[END_REF][START_REF] Schiavi | Unidirectional opioid-cannabinoid cross-tolerance in the modulation of social play behavior in rats[END_REF]. Hence, activating the physiological functions of the ECS seems to induce promoting effects on social behaviors.

In line with the these results, the blockade of CB1r signaling is almost universally recognized as an « anti-social » treatment in animal research (Table 3), and could therefore be instrumental in inducing ASDlike phenotypes in WT animals with unaltered endocannabinoid tone (in contrast to genetic mouse models of ASD, e.g., the Fmr1-KO and BTBR lines, where normalization of ECS alterations through CB1 inhibition resulted in rescuing of certain ASD-like phenotypes, as described before). Systemic administration of the CB1r blocker AM251 in adult male capuchin monkeys induced a decrease in social and an increase in Pietropaolo and G. Marsicano Neuroscience and Biobehavioral Reviews xxx (xxxx) 1-14 self-directed behaviors, as well as resistance to aversive memory extinction, without concomitant changes in the subjects' locomotor activity [START_REF] Gonczarowska | CB1 receptor antagonism in capuchin monkeys alters social interaction and aversive memory extinction[END_REF]. AM251 was shown to decrease social interest also in rats [START_REF] Seillier | Phencyclidine-induced social withdrawal results from deficient stimulation of cannabinoid CB(1) receptors: implications for schizophrenia[END_REF] and mice [START_REF] Umathe | Endocannabinoids mediate anxiolytic-like effect of acetaminophen via CB1 receptors[END_REF][START_REF] Wei | Endocannabinoid signaling mediates oxytocin-driven social reward[END_REF], where it also altered social memory [START_REF] Litvin | CB1 receptor signaling regulates social anxiety and memory[END_REF]. In line with these studies, we have investigated the ability of the CB1r antagonist rimonabant of inducing ASD-like behaviors in wild type B6 mice, i.e., the background that is mostly used to engineer ASD models, including the Fmr1-KO mouse. We demonstrated that an acute injection of increasing doses of rimonabant reduced the levels of affiliative behaviors towards a female, in particular of anogenital sniffings, and the number of ultrasonic vocalizations, while it increased those of self-grooming, i.e., it induced an ASD-like phenotype similar to that observed in Fmr1-KO mice [START_REF] Pietropaolo | The role of the endocannabinoid system in autism spectrum disorders: evidence from mouse studies[END_REF]. Interestingly, no effect on social behaviors was detected following administration of selective CB2 inhibitors [START_REF] Argue | Activation of both CB1 and CB2 endocannabinoid receptors is critical for masculinization of the developing medial amygdala and juvenile social play behavior[END_REF][START_REF] Trezza | Divergent effects of anandamide transporter inhibitors with different target selectivity on social play behavior in adolescent rats[END_REF], suggesting an exclusive role of CB1 receptor in modulating social interactions.

U N C O R R E C T E D P R O O F S.

Behavioral effects of genetic targeting the ECS in WT mice

Interestingly, ASD-like behaviors were also described following the genetic deletion of CB1r, thus suggesting the possible relevance of CB1r-KO mice to model ASD (Table 3). Impaired sociability [START_REF] Haller | CB1 cannabinoid receptors mediate anxiolytic effects: convergent genetic and pharmacological evidence with CB1specific agents[END_REF] and social exploration [START_REF] Haring | Cannabinoid type-1 receptor signaling in central serotonergic neurons regulates anxiety-like behavior and sociability[END_REF][START_REF] Haring | Circuit specific functions of cannabinoid CB1 receptor in the balance of investigatory drive and exploration[END_REF] was observed in these KOs [although the effects vary according with the testing context [START_REF] Gould | Acetaminophen differentially enhances social behavior and cortical cannabinoid levels in inbred mice[END_REF] and the type of social stimulus [START_REF] Terzian | Cannabinoid receptor type 1 receptors on GABAergic vs. glutamatergic neurons differentially gate sex-dependent social interest in mice[END_REF]], as well as altered social memory [START_REF] Litvin | CB1 receptor signaling regulates social anxiety and memory[END_REF]. While repetitive behaviors, such as enhanced levels of self-grooming, were observed in CB1r-KOs [START_REF] Litvin | CB1 receptor signaling regulates social anxiety and memory[END_REF][START_REF] Terzian | The dopamine and cannabinoid interaction in the modulation of emotions and cognition: assessing the role of cannabinoid CB1 receptor in neurons expressing dopamine D1 receptors[END_REF], we have recently described qualitative and quantitative alterations in ultrasonic vocalizations in CB1r-KO mice, both during development (i.e., in the first post-natal week) and at adulthood [START_REF] Fyke | Communication and social interaction in the cannabinoid-type 1 receptor null mouse: implications for autism spectrum disorder[END_REF]. These abnormalities were detected through an extensive spectrographic analysis of the calls, including multiple testing days and qualitative parameters. CB1r-KO mice showed also a lack of social interest in the 3chamber test at adulthood, as well as reduced social investigation in the direct interaction test [START_REF] Fyke | Communication and social interaction in the cannabinoid-type 1 receptor null mouse: implications for autism spectrum disorder[END_REF]. The emerging importance of CB1r-KO mice to study the role of the ECS in ASD is remarkable, especially because of the availability of KOs in specific neuronal populations, helping to disentangle the impact of glutamatergic, gabaergic, serotonergic, dopaminergic or glial pools of these receptors in the modulation of social and ASD-relevant behaviors [START_REF] Haring | Cannabinoid type-1 receptor signaling in central serotonergic neurons regulates anxiety-like behavior and sociability[END_REF][START_REF] Haring | Circuit specific functions of cannabinoid CB1 receptor in the balance of investigatory drive and exploration[END_REF][START_REF] Litvin | CB1 receptor signaling regulates social anxiety and memory[END_REF][START_REF] Terzian | The dopamine and cannabinoid interaction in the modulation of emotions and cognition: assessing the role of cannabinoid CB1 receptor in neurons expressing dopamine D1 receptors[END_REF][START_REF] Terzian | Cannabinoid receptor type 1 receptors on GABAergic vs. glutamatergic neurons differentially gate sex-dependent social interest in mice[END_REF].

In line with the pharmacological findings summarized before, the genetic deletion of FAAH or DAGL induced respectively pro-social or ASD-like behavioral phenotypes [START_REF] Cassano | Evaluation of the emotional phenotype and serotonergic neurotransmission of fatty acid amide hydrolase-deficient mice[END_REF][START_REF] Shonesy | Role of striatal direct pathway 2arachidonoylglycerol signaling in sociability and repetitive behavior[END_REF][START_REF] Wei | Endocannabinoid signaling mediates oxytocin-driven social reward[END_REF], thus confirming the critical role of the ECs. Interestingly, the pro-social phenotype of FAAH-knock-out mice was accompanied by increased serotonergic tone in cortical areas and both elements were eliminated by the acute administration of rimonabant, thus suggesting the critical relevance of anandamide-mediated hyperactivation of CB1r [START_REF] Cassano | Evaluation of the emotional phenotype and serotonergic neurotransmission of fatty acid amide hydrolase-deficient mice[END_REF].

Conclusions and perspectives

Clinical and pre-clinical data strongly support the involvement of the ECS in the etiopathology of ASD, and its relevance as a therapeutic target to design novel pharmacological treatments. Evidence from mouse models of ASD and pharmacological manipulations of the ECS in wild type animals highlight the importance of an imbalance in ECS signaling as a common potential etiopathological mechanism of this complex disorder.

The ECS could therefore be considered as a good candidate to fit into the theory of ASD of "multiple genes, common pathways", also be-cause of its major modulatory role of neuronal functions and brain development. In this latter respect, some authors have recently suggested that the impact of the ECS in ASD could be specifically related to its modulation of brain developmental processes [START_REF] Yeh | Perspectives on the role of endocannabinoids in autism spectrum disorders[END_REF], an issue that has been poorly elucidated in the context of ASD models. The use of induced pluripotent stem cell derived neurons has already provided valuable insight into the pathogenesis of neurological disorders such as ASDs [START_REF] Wen | Synaptic dysregulation in a human iPS cell model of mental disorders[END_REF], and should therefore be applied to future studies to try to differentiate the relevance of the ECS in abnormal brain development from mature synaptic functionality [START_REF] Yeh | Perspectives on the role of endocannabinoids in autism spectrum disorders[END_REF].

The complexity characterizing ASD etiopathology reflects somehow the complexity of the ECS itself. On one hand, the discovery of the relevance of endocannabinoids in ASD raised new possibilities for safe targeting the ECS to treat this disorder, as demonstrated by the evidence reviewed here supporting the therapeutic use of phytocannabinoids, such as CBD and CBDV. These compounds are promising because of their anti-inflammatory properties, and because they can enhance endocannabinoid levels without the adverse effects of CB1r direct modulation. On the other hand, our knowledge of the complexity of the ECS has further increased, complicating clinical approaches. The discovery that endocannabinoids can activate different novel receptors and that their metabolic pathways are often shared with other mediators has led to the inclusion of the ECS into an expanded signaling system, known as the endocannabinoidome [START_REF] Cristino | Cannabinoids and the expanded endocannabinoid system in neurological disorders[END_REF]. This enlarged ECS, including several mediators that are biochemically related to the endocannabinoids, together with their receptors and metabolic enzymes, is likely to provide novel molecular targets of therapeutic relevance to be tested in future studies on ASD [START_REF] Cristino | Cannabinoids and the expanded endocannabinoid system in neurological disorders[END_REF].

Finally, the studies reviewed here support the central relevance of behavioral analysis in modern neuroscientific research. Indeed, the investigation of the role of the ECS as in ASD pathology may be considered itself as a powerful example of the reasons why behavior matters to brain science. This is evident from the need of a thorough behavioral characterization of mouse models of ASD both to demonstrate the therapeutic effects of pharmacological treatments targeting the ECS and to evaluate the ability of manipulations of the ECS to induce ASD-like phenotypes. This need is due to the lack of biomarkers for ASD other than behavioral alterations, and to the complexity of the behavioral ASDrelevant phenotypes. Furthermore, research on the ECS and ASD can provide with an opportunity to further develop behavioral science itself, as demonstrated by the advanced behavioral approaches used in some studies reviewed here to investigate the role of the ECS in modulating complex social behaviors in mice, e.g., ultrasonic communication. The strong complexity of ASD symptomatology affecting highly developed social domains represents indeed a challenge for behavioral neuroscience, thus contributing to assign a centrality to behavioral analysis in the present and future neuroscientific research.

U N C O R R E C T E D P R O O F

is supported by INSERM, European Research Council (Endofood, ERC-2010-StG-260515 and CannaPreg, ERC-2014-PoC-640923, Mi-CaBra, ERC-2017-AdG-786467), Fondation pour la Recherche Medicale (FRM, DRM20101220445), the Human Frontiers Science Program, Region Nouvelle Aquitaine, Agence Nationale de la Recherche (ANR, Neu-roNutriSens ANR-13-BSV4-0006; ORUPS ANR-16-CE37-0010-01; Ca-CoVi ANR 18-CE16-0001-02; MitObesity ANR 18-CE14-0029-01; BRAIN ANR-10-LABX-0043). The authors are grateful to Luigi Bellocchio for critical reading and reviewing of the manuscript.

  . FAAH: fatty acid amide hydrolase. MAGL: monoacyl-glycerol lipase. ECs: endocannabinoids. DAGL-a: Diacylglycerol lipase alpha. EPM: elevated plus maze. 3-COMP: three compartment test. USVs: ultrasonic vocalizations. SI: social interaction. ↑increase. ↓decrease. ↔no change.

Fig. 1 .

 1 Fig. 1. Schematic representation of ECS functionality and its alterations in patients with ASD. ECs: endocannabinoids. AEA: anandamide. 2-AG: 2-arachidonoyl-glycerol. NAPE-PLD: N-acyl phosphatidylethanolamine-specific phospholipase D. DAGL-a: Diacylglycerol lipase alpha. FAAH: fatty acid amide hydrolase. MAGL: monoacyl-glycerol lipase. The elements of the system that are known to be altered in ASD patients are marked in red color. Direction of the red arrows indicates increase or decrease.deletion of the gene encoding the CB1r[START_REF] Bellocchio | Bimodal control of stimulated food intake by the endocannabinoid system[END_REF][START_REF] Bellocchio | Activation of the sympathetic nervous system mediates hypophagic and anxiety-like effects of CB(1) receptor blockade[END_REF][START_REF] Monory | The endocannabinoid system controls key epileptogenic circuits in the hippocampus[END_REF], as well as the genes of associated metabolic proteins [NAPE-PLD, DAGL-a, FAAH and MAGL; reviewed in (Busquets-[START_REF] Busquets-Garcia | CB1 receptor signaling in the brain: extracting specificity from ubiquity[END_REF]] are available (see also Table3). These genetically

Table 1

 1 Evidence for ECS alterations in animal models of ASD.

	Modelling approach	Model name Description of ECS alteration	References
	Monogenic associated syndromes Strain differences Candidate gene Gestational exposure to environmental insults (e.g., teratogens, viral/bacterial infection)	Fmr1-KO mice BTBR mice NLGN3-R451C knockin and NLGN3 knockout mice VPA rats LPS rats	↔ CB1r expression and 2-AG levels ↓ EC-mediated LTD at excitatory synapses in forebrain ↑ MAGL activity in the striatum ↓ EC-mediated LTD at inhibitory synapses in hippocampus and striatum ↑ CB1r binding and GTP-gamma activation ↑CB2 gene expression in the cerebellum ↓ tonic EC signalling in the hippocampus, cortex, amygdala and dorsal striatum ↔ tonic EC signalling and mobilization in the nucleus accumbens ↓ CB1r phosphorylation in the amygdala, hippocampus and dorsal striatum ↓ gene expression of DGL-α in the cerebellum ↓NAPE PLD and ↑FAAH in the whole brain ↓CB1, ↑ FAAH in the amygdala	(Zhang and Alger, 2010; Jung et al., 2012) (Jung et al., 2012) (Maccarrone et al., 2010) (Maccarrone et al., 2010; Zhang and Alger, 2010) (Gould et al., 2012, 2014) (Liu et al., 2009) (Foldy et al., 2013; Speed et al., 2015; Hosie et al., 2018; Martella et al., 2018) (Rothwell et al., 2015) (Servadio et al., 2016) (Servadio et al., 2016) (Servadio et al., 2016) (Doenni et al., 2016)

NLG: neurolegin. VPA: valproic acid. LPS: lipopolysaccharide. LTD: long-term depression. ECs: endocannabinoids. 2-AG: 2-arachidonoyl-glycerol. NAPE-PLD: N-acyl phosphatidylethanolamine-specific phospholipase D. DAGL-a: Diacylglycerol lipase alpha. FAAH: fatty acid amide hydrolase. MAGL: monoacylglycerol lipase. ↑increase. ↓decrease. ↔no change.

Table 2

 2 Pharmacological therapies targeting the ECS in animal models of ASD.

	Treatment action on ECS	Drug	Dose	ASD model	Therapeutic effects	Behavioral tests	References
	CB2 antagonist CB1 antagonist CB1-agonist ACEA AM630 SR141716A 1 mg/kg; 1 mg/kg; acute acute 0,03-1 mg/kg; acute/chronic AM251 2 mg/Kg 10µM, in bath application 20µM, perfusion Win 55,212-2 0,3 or 1 mg/kg; acute FAAH-inhibitor URB597 0,3 mg/kg 0,3 mg/kg 0,3 mg/kg and 1 mg/Kg 0.05 mg/Kg acute PF-3845 10 mg/Kg acute PF-04457845 1 mg/Kg acute MAGL-inhibitor JZL-184 16 mg/kg	Fmr1-KO mouse Fmr1-KO mouse Fmr1-KO mouse BTBR mouse NLGN-3 mouse NLGN-3 mouse NLGN-3 mouse Fmr1-KO mouse Fmr1-KO mouse BTBR mouse VPA-rat VPA-rat LPS-rat Fmr1-KO mouse	rescue of anxiety alterations and audiogenic seizures rescue of cognitive deficits, audiogenic seizures, altered nociception and abnormal mTOR signaling rescue of cognitive deficits no effect on sociability deficits rescue of increased inhibitory transmission in somatosensory cortex rescue of alterations in tonic EC signalling in the dorsal striatum _ EPM NOR NOR 3 comp _ Rescue of enhanced aggressiveness resident-intruder amelioration of cognitive deficits, no effect on anxiety PA, EPM rescue of sociability deficits 3 comp rescue of sociability deficits 3 comp rescue of communication and social deficits at infancy, adolescence and adulthood. Elimination of anxiety and stereotypic behaviors USVs, homing, hole board, social play, 3 comp, EPM rescue of social deficits 3 comp rescue of social deficits direct SI no effect PA normalization of hyperactivity and altered anxiety EPM, OF	(Busquets-Garcia et al., 2013) (Busquets-Garcia et al., 2013) (Busquets-Garcia et al., 2013; Gomis-Gonzalez et al., 2016) (Wei et al., 2016) (Speed et al., 2015) (Martella et al., 2018) (Hosie et al., 2018) (Qin et al., 2015) (Wei et al., 2016) (Wei et al., 2016) (Servadio et al., 2016) (Kerr et al., 2013) (Doenni et al., 2016) (Qin et al., 2015) (Jung et al., 2012)

FAAH: fatty acid amide hydrolase. MAGL: monoacyl-glycerol lipase. NLG: neurolegin. VPA: valproic acid. LPS: lipopolysaccharide. EPM: elevated plus maze. OF: open field. 3-COMP: three compartment test. NOR: novel object recognition. PA: passive avoidance. USVs: ultrasonic vocalizations. SI: social interaction.

Table 3

 3 ASD-like phenotypes induced by pharmacological and genetic targeting of the ECS in rodents.

	Action on ECS	GT	Drug	Dose	ASD-like behavioral phenotype	Behavioral test	References
	CB1-antagonist CB2-antagonist FAAH-inhibitor MAGL-inhibitor CB1-agonist WT WT mice WT rats AM630 AM251 SR141716A 3 or 10 mg/Kg, 2 mg/Kg, acute 5 mg/Kg, acute acute 1 mg/Kg, subchronic ↔ social interaction ↓ sociability, ↔ anxiety ↓ social interaction and social memory ↓ social interaction and USV emission,↑ repetitive behavior SR144528 0.1 mg/Kg, acute ↔ social interaction WT mice URB597 1 mg/Kg, acute ↔ sociability or anxiety WT rats URB597 0.01 mg/Kg, acute ↑ social interaction 0.1 mg/Kg, acute ↑ social interaction WT JZL-184 8 mg/Kg, acute ↑ social interaction mice WT rats JZL-184 1 mg/Kg, acute ↑ social interaction mice Win 55,212-2 0.4 mg/Kg, acute; ↓ social interaction 0.1 mg/Kg, chronic 0.1 mg/Kg, acute ↓ social interaction WT rats Win 55,212-2 0.1 -0.3 mg/Kg, ↑ social interaction acute WT mice CP 55,940 0.1 mg/Kg, acute ↔ social interaction	3-comp, EPM SI SI SI SI 3 comp, EPM SI SI SI SI SI 3-COMP SI SI	(Wei et al., 2016) (Litvin et al., 2013; Umathe et al., 2009) (Pietropaolo et al., 2020; Terzian et al., 2014) (Argue et al., 2017) (Trezza and Vandershuren 2009) (Wei et al., 2016) (Manduca et al., 2014) (Trezza et al., 2012; Trezza and Vandershuren 2008) (Manduca et al., 2016) (Schiavi et al., 2019) (Pietropaolo et al., 2015) (Gould et al., 2012) (Almeida et al., 2014) (Tambaro et al., 2013)
	CB1-deletion	CB1-KO mice			↓ sociability and social exploration	SI, 3-COMP

WT rats ACEA 0.1 -1 mg/Kg, acute ↔ social interaction SI

[START_REF] Almeida | Effects of cannabinoid and vanilloid drugs on positive and negative-like symptoms on an animal model of schizophrenia: the SHR strain[END_REF][START_REF] Argue | Activation of both CB1 and CB2 endocannabinoid receptors is critical for masculinization of the developing medial amygdala and juvenile social play behavior[END_REF]

Acknowledgements

This paper is dedicated to the memory of our friend and colleague Federico Massa.

S. Pietropaolo received funding from CNRS and Bordeaux University, from GW Research Limited (Cambridge, UK; GWR-01834-DIS-ARA-02034; 2017-2019), Association "Autour de Williams" (Paris, France) and "Fondation pour l'audition" (Paris, France). G. Marsicano

Data availability

No data was used for the research described in the article.

Declaration of Competing Interest

The authors report no declarations of interest.