Caterina Michetti

Chronic and Acute Intranasal Oxytocin Produce Divergent Social Effects in Mice

Intranasal administration of oxytocin (OXT) might be a promising new adjunctive therapy for mental disorders characterized by social behavioral alterations such as autism and schizophrenia. Despite promising initial studies in humans, it is not yet clear the specificity of the behavioral effects induced by chronic intranasal OXT and if chronic intranasal OXT could have different effects compared with single administration. This is critical for the aforementioned chronic mental disorders that might potentially involve life-long treatments. As a first step to address these issues, here we report that chronic intranasal OXT treatment in wild-type C57BL/6J adult mice produced a selective reduction of social behaviors concomitant to a reduction of the OXT receptors throughout the brain. Conversely, acute intranasal OXT treatment produced partial increases in social behaviors towards opposite-sex novel-stimulus female mice, while on the other hand, it decreased social exploration of same-sex novel stimulus male mice, without affecting social behavior towards familiar stimulus male mice. Finally, prolonged exposure to intranasal OXT treatments did not alter, in wild-type animals, parameters of general health such as body weight, locomotor activity, olfactory and auditory functions, nor parameters of memory and sensorimotor gating abilities. These results indicate that a prolonged over-stimulation of a ‘healthy’ oxytocinergic brain system, with no inherent deficits in social interaction and normal endogenous levels of OXT, results in specific detrimental effects in social behaviors.

Characterization of Neonatal Vocal and Motor Repertoire of Reelin Mutant Mice

Reelin is a large secreted extracellular matrix glycoprotein playing an important role in early neurodevelopment. Several genetic studies found an association between RELN gene and increased risk of autism suggesting that reelin deficiency may be a vulnerability factor in its etiology. Moreover, a reduced reelin expression has been observed in several brain regions of subjects with Autism Spectrum Disorders. Since a number of reports have documented presence of vocal and neuromotor abnormalities in patients with autism and suggested that these dysfunctions predate the onset of the syndrome, we performed a fine-grain characterization of the neonatal vocal and motor repertoire in reelin mutant mice to explore the developmental precursors of the disorder. Our findings evidence a general delay in motor and vocal development in heterozygous (50% reduced reelin) and reeler (lacking reelin gene) mutant mice. As a whole, an increased number of calls characterized heterozygous pups emission. Furthermore, the typical ontogenetic peak in the number of calls characterizing wild-type pups on postnatal day 4 appeared slightly delayed in heterozygous pups (to day 6) and was quite absent in reeler littermates, which exhibited a flat profile during development. We also detected a preferential use of a specific call category (two-components) by heterozygous and reeler mice at postnatal days 6 and 8 as compared to their wild-type littermates. With regard to the analysis of spontaneous movements, a differential profile emerged early in development among the three genotypes. While only slight coordination difficulties are exhibited by heterozygous pups, all indices of motor development appear delayed in reeler mice. Overall, our results evidence a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reelin mutant pups.

The chromatin remodeling factor CHD7 controls cerebellar development by regulating reelin expression

The mechanisms underlying the neurodevelopmental deficits associated with CHARGE syndrome, which include cerebellar hypoplasia, developmental delay, coordination problems, and autistic features, have not been identified. CHARGE syndrome has been associated with mutations in the gene encoding the ATP-dependent chromatin remodeler CHD7. CHD7 is expressed in neural stem and progenitor cells, but its role in neurogenesis during brain development remains unknown. Here we have shown that deletion of Chd7 from cerebellar granule cell progenitors (GCps) results in reduced GCp proliferation, cerebellar hypoplasia, developmental delay, and motor deficits in mice. Genome-wide expression profiling revealed downregulated expression of the gene encoding the glycoprotein reelin (Reln) in Chd7-deficient GCps. Recessive RELN mutations have been associated with severe cerebellar hypoplasia in humans. We found molecular and genetic evidence that reductions in Reln expression contribute to GCp proliferative defects and cerebellar hypoplasia in GCp-specific Chd7 mouse mutants. Finally, we showed that CHD7 is necessary for maintaining an open, accessible chromatin state at the Reln locus. Taken together, this study shows that Reln gene expression is regulated by chromatin remodeling, identifies CHD7 as a previously unrecognized upstream regulator of Reln, and provides direct in vivo evidence that a mammalian CHD protein can control brain development by modulating chromatin accessibility in neuronal progenitors.

Modeling Social Communication Deficits in Mouse Models of Autism

Male and female mice emit ultrasonic vocalizations during infancy when pups are separated from mother and littermates, as well as at adulthood in different experimental/social contexts. Mouse ultrasonic vocalizations had become now a popular assay for behavioral phenotyping throughout the life-span of models of autism since this response represents the best option to detect deficits within the social communication domain in the mouse species. In the present review, we describe the available methods to elicit and record mouse ultrasonic vocalizations in different social contexts and at different ages. Behavioral data collected on autism animal models in these paradigms/ contexts are also discussed. Moreover, we strongly emphasized the need of a standardization of the behavioral methods to better compare results from different laboratories. Thanks to the progresses of computer technology, researchers can now perform detailed analyses of the vocal repertoire (classifying ultrasonic vocalizations into different categories) in autism mouse models. Recently, these analyses have revealed unusual vocal patterns in selected mouse lines. This innovative approach allows to detect also qualitative alterations in the social communication repertoire usually not identified with the standard analysis of emission rate. Future studies should be aimed at performing quantitative and qualitative analyses of vocalization patterns also in preclinical studies evaluating potential treatments in validated autism mouse models.

The PRRT2 knockout mouse recapitulates the neurological diseases associated with PRRT2 mutations

Heterozygous and rare homozygous mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia episodic ataxia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function. Recently, an important role for PRTT2 in the neurotransmitter release machinery, brain development and synapse formation has been uncovered. In this work, we have characterized the phenotype of a mouse in which the PRRT2 gene has been constitutively inactivated (PRRT2 KO). β-galactosidase staining allowed to map the regional expression of PRRT2 that was more intense in the cerebellum, hindbrain and spinal cord, while it was localized to restricted areas in the forebrain. PRRT2 KO mice are normal at birth, but display paroxysmal movements at the onset of locomotion that persist in the adulthood. In addition, adult PRRT2 KO mice present abnormal motor behaviors characterized by wild running and jumping in response to audiogenic stimuli that are ineffective in wild type mice and an increased sensitivity to the convulsive effects of pentylentetrazol. Patch-clamp electrophysiology in hippocampal and cerebellar slices revealed specific effects in the cerebellum, where PRRT2 is highly expressed, consisting in a higher excitatory strength at parallel fiber-Purkinje cell synapses during high frequency stimulation. The results show that the PRRT2 KO mouse reproduces the motor paroxysms present in the human PRRT2-linked pathology and can be proposed as an experimental model for the study of the pathogenesis of the disease as well as for testing personalized therapeutic approaches.

Ultrasonic vocalization in rats self-administering heroin and cocaine in different settings: evidence of substance-specific interactions between drug and setting

RationaleClinical and preclinical evidence indicates that the setting of drug use affects drug reward in a substance-specific manner. Heroin and cocaine co-abusers, for example, indicated distinct settings for the two drugs: heroin being used preferentially at home and cocaine preferentially outside the home. Similar results were obtained in rats that were given the opportunity to self-administer intravenously both heroin and cocaine.ObjectivesThe goal of the present study was to investigate the possibility that the positive affective state induced by cocaine is enhanced when the drug is taken at home relative to a non-home environment, and vice versa for heroin.MethodsTo test this hypothesis, we trained male rats to self-administer both heroin and cocaine on alternate days and simultaneously recorded the emission of ultrasonic vocalizations (USVs), as it has been reported that rats emit 50-kHz USVs when exposed to rewarding stimuli, suggesting that these USVs reflect positive affective states.ResultsWe found that Non-Resident rats emitted more 50-kHz USVs when they self-administered cocaine than when self-administered heroin whereas Resident rats emitted more 50-kHz USVs when self-administering heroin than when self-administering cocaine. Differences in USVs in Non-Resident rats were more pronounced during the first self-administration (SA) session, when the SA chambers were completely novel to them. In contrast, the differences in USVs in Resident rats were more pronounced during the last SA sessions.ConclusionThese findings indicate that the setting of drug taking exerts a substance-specific influence on the ability of drugs to induce positive affective states.

Altered Neocortical Gene Expression, Brain Overgrowth and Functional Over-Connectivity in Chd8 Haploinsufficient Mice

Abstract Truncating CHD8 mutations are amongst the highest confidence risk factors for autism spectrum disorder (ASD) identified to date. Here, we report that Chd8 heterozygous mice display increased brain size, motor delay, hypertelorism, pronounced hypoactivity, and anomalous responses to social stimuli. Whereas gene expression in the neocortex is only mildly affected at midgestation, over 600 genes are differentially expressed in the early postnatal neocortex. Genes involved in cell adhesion and axon guidance are particularly prominent amongst the downregulated transcripts. Resting-state functional MRI identified increased synchronized activity in cortico-hippocampal and auditory-parietal networks in Chd8 heterozygous mutant mice, implicating altered connectivity as a potential mechanism underlying the behavioral phenotypes. Together, these data suggest that altered brain growth and diminished expression of important neurodevelopmental genes that regulate long-range brain wiring are followed by distinctive anomalies in functional brain connectivity in Chd8+/− mice. Human imaging studies have reported altered functional connectivity in ASD patients, with long-range under-connectivity seemingly more frequent. Our data suggest that CHD8 haploinsufficiency represents a specific subtype of ASD where neuropsychiatric symptoms are underpinned by long-range over-connectivity.

PRRT2, a network stability gene

Specific paroxysmal disorders, namely benign infantile epilepsy (BFIE), kinesigenic dyskinesia (PKD), infantile convulsions and choreoathetosis (ICCA) and hemiplegic migraine (HM), are associated with mutations in the gene encoding for PRoline-Rich Transmembrane protein 2 (PRRT2; Figure 1A, 1B). PRRT2 is a neuronspecific protein expressed on neuronal membranes and at synapses, with a prevalent presynaptic location [1, 2]. Several nonsense, missense and frame-shift mutations were identified in the PRRT2 gene, but the vast majority of patients (80%) carry the same frameshift single-nucleotide duplication (c.649dupC) that leads to a premature stop codon and results in a loss-of-function pathogenetic mechanism [3]. Despite the extensive characterization of PRRT2 mutations, no clear evidence for genotypephenotype correlation exists and the three main diseases (BFIE, PKD and PKD/ICCA) form a continuous spectrum, starting from BFIE in the first months of life and evolving to PKD or PKD/ICCA often in association with HM during adolescence [4-5]. To model the disorders and investigate the underlying neurobiological alterations, we recently characterized a PRRT2 knock out mouse (PRRT2 KO; Figure 1C), carrying a constitutive inactivation of the PRRT2 gene [6]. The promoterless lacZ gene integrated into the PRRT2 locus allowed mapping the PRRT2 regional expression, that is not widespread, but rather concentrated at restricted brain areas. Interestingly, the expression is high in neurons of the lower hindbrain particularly in the cerebellum, a brain area involved in the generation of motor/epileptic phenotype and in which altered synaptic plasticity at the parallel fibersPurkinje cells synapse was found [6] (Figure 1D). A selective staining was also identified in the cerebral cortex, claustrum and dorsal horns of the spinal cord. Moreover, in the hippocampus, PRRT2 is particularly expressed in the hilus of the dentate gyrus, where mossy cells control the excitability of granule cells, playing a role in preventing hippocampal seizures (Figure 1D). Notably, all the brain regions positive for PRRT2 are involved in processing sensory information, motor disorders and epilepsy, all neurological traits present in patients with PRRT2 mutations. The PRRT2 KO mouse recapitulates many of the phenotypic features of the human PRRT2-linked disorders, showing abnormal motor behaviors and a motor/epileptic phenotype in response to environmental stimuli [6]. The motor problems are represented by gait abnormalities and a peculiar paroxysmal backwalking appearing early in the postnatal life and persisting in the adult [4]. The motor/ epileptic phenotype of PRRT2 KO mice becomes dramatic in response to audiogenic stimuli (Figure 1E), which trigger wild running, backwalking and jumping, and to the administration of convulsants, such as pentylentetrazole (Figure 1F). In both kinds of provocations, however, the seizure propensity was not very severe, consistent with the mild epileptic phenotype of patients bearing PRRT2 mutations [4, 5]. Overall, the PRRT2 KO mouse reproduces the paroxysmal traits described in PRRT2 patients and characterized by sudden and short attacks that usually occur periodically in response to a sensory trigger. The episodic nature of these events suggests that they result from network instability caused by changes in the excitation/inhibition balance. Interestingly, data obtained in primary neurons showed that PRRT2 silencing is associated with alterations of synaptic transmission and short-term plasticity [1]. PRRT2 silencing gives rise to a decreased number of excitatory synapses and a marked impairment of fast synchronous neurotransmitter release. These effects are associated with a decrease in Ca2+ sensitivity and release probability, revealing a role for Editorial

The Knockout of Synapsin II in Mice Impairs Social Behavior and Functional Connectivity Generating an ASD-like Phenotype

Autism spectrum disorders (ASD) and epilepsy are neurodevelopmental conditions that appear with high rate of co-occurrence, suggesting the possibility of a common genetic basis. Mutations in Synapsin (SYN) genes, particularly SYN1 and SYN2, have been recently associated with ASD and epilepsy in humans. Accordingly, mice lacking Syn1 or Syn2, but not Syn3, experience epileptic seizures and display autistic-like traits that precede the onset of seizures. Here, we analyzed social behavior and ultrasonic vocalizations emitted in 2 social contexts by SynI, SynII, or SynIII mutants and show that SynII mutants display the most severe ASD-like phenotype. We also show that the behavioral SynII phenotype correlates with a significant decrease in auditory and hippocampal functional connectivity as measured with resting state functional magnetic resonance imaging (rsfMRI). Taken together, our results reveal a permissive contribution of Syn2 to the expression of normal socio-communicative behavior, and suggest that Syn2-mediated synaptic dysfunction can lead to ASD-like behavior through dysregulation of cortical connectivity.

Rodent Vocalization Studies in Animal Models of the Autism Spectrum Disorder

Abstract In preclinical rodent models of autism spectrum disorders (ASD), the study of ultrasonic vocalizations (USVs) has provided the opportunity to evaluate in ethologically appropriate contexts a behavioral domain (social communication) that is relevant to the core diagnostic of the ASD symptomatology. In this chapter, we review a selection of data concerning USVs in mouse models of ASD both in the neonatal phase and in adulthood in different experimental contexts and with the emphasis on the spectrographic analyses to assess the role of vocalizations in these models. Abnormalities have been evidenced both quantitatively (calling rate) and qualitatively (range of the vocal repertoire). We also highlight several promises and a few caveats in the use of USVs for behavioral phenotyping of ASD mouse models, with some suggestions to maximize the translational potential of these studies.