Cannabidiol and positive effects on object recognition memory in an in vivo model of Fragile X Syndrome: Obligatory role of hippocampal GPR55 receptors.

Cannabidiol (CBD), a non-psychotomimetic constituent of Cannabis sativa, has been recently approved for epileptic syndromes often associated with Autism spectrum disorder (ASD). However, the putative efficacy and mechanism of action of CBD in patients suffering from ASD and related comorbidities remain debated, especially because of the complex pharmacology of CBD. We used pharmacological, immunohistochemical and biochemical approaches to investigate the effects and mechanisms of action of CBD in the recently validated Fmr1-Δexon 8 rat model of ASD, that is also a model of Fragile X Syndrome (FXS), the leading monogenic cause of autism. CBD rescued the cognitive deficits displayed by juvenile Fmr1-Δexon 8 animals, without inducing tolerance after repeated administration. Blockade of CA1 hippocampal GPR55 receptors prevented the beneficial effect of both CBD and the fatty acid amide hydrolase (FAAH) inhibitor URB597 in the short-term recognition memory deficits displayed by Fmr1-Δexon 8 rats. Thus, CBD may exert its beneficial effects through CA1 hippocampal GPR55 receptors. Docking analysis further confirmed that the mechanism of action of CBD might involve competition for brain fatty acid binding proteins (FABPs) that deliver anandamide and related bioactive lipids to their catabolic enzyme FAAH. These findings demonstrate that CBD reduced cognitive deficits in a rat model of FXS and provide initial mechanistic insights into its therapeutic potential in neurodevelopmental disorders.

Fatty Acid Amide Hydrolase and Cannabinoid Receptor Type 1 Genes Regulation is Modulated by Social Isolation in Rats.

Social isolation is a state of lack of social connections, involving the modulation of different molecular signalling cascades and associated with high risk of mental health issues. To investigate if and how gene expression is modulated by social experience at the central level, we analyzed the effects of 5 weeks of social isolation in rats focusing on endocannabinoid system genes transcription in key brain regions involved in emotional control. We observed selective reduction in mRNA levels for fatty acid amide hydrolase (Faah) and cannabinoid receptor type 1 (Cnr1) genes in the amygdala complex and of Cnr1 in the prefrontal cortex of socially isolated rats when compared to controls, and these changes appear to be partially driven by trimethylation of Lysine 27 and acetylation of Lysine 9 at Histone 3. The alterations of Cnr1 transcriptional regulation result also directly correlated with those of oxytocin receptor gene. We here suggest that to counteract the effects of SI, it is of relevance to restore the endocannabinoid system homeostasis via the use of environmental triggers able to revert those epigenetic mechanisms accounting for the alterations observed.

Group I and group II metabotropic glutamate receptors are upregulated in the synapses of infant rats prenatally exposed to valproic acid.

RATIONALE: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interaction and restricted/stereotyped behavior. Prenatal exposure to valproic acid (VPA) is associated with an increased risk of developing ASD in humans and autistic-like behaviors in rodents. Increasing evidence indicates that dysfunctions of glutamate receptors at synapses are associated with ASD. In the VPA rat model, an involvement of glutamate receptors in autism-like phenotypes has been suggested; however, few studies were carried out on metabotropic glutamate (mGlu) receptors. OBJECTIVES: We examined the protein expression levels of group I (mGlu1 and mGlu5) and group II (mGlu2/3) mGlu receptors in rats prenatally exposed to VPA and evaluated the effect of mGlu receptor modulation on an early autism-like phenotype in these animals. METHODS: We used western blotting analysis on synaptosomes obtained from forebrain of control and VPA rats at different ages (postnatal day P13, 35, 90) and carried out ultrasonic vocalization (USV) emission test in infant control and VPA rats. RESULTS: The expression levels of all these receptors were significantly increased in infant VPA rats. No changes were detected in adolescent and adult rats. An acute treatment with the preferential mGlu2/3 antagonist, LY341495, attenuated the impairment in the USV emission in VPA rats. No effect was observed after a treatment with the mGlu5 selective antagonist, MTEP. CONCLUSIONS: Our findings demonstrate that the expression of group I and group II mGlu receptors is upregulated at synapses of infant VPA rats and suggest that mGlu2/3 receptor modulation may have a therapeutic potential in ASD.

Maternal Immune Activation Induced by Prenatal Lipopolysaccharide Exposure Leads to Long-Lasting Autistic-like Social, Cognitive and Immune Alterations in Male Wistar Rats.

Several studies have supported the association between maternal immune activation (MIA) caused by exposure to pathogens or inflammation during critical periods of gestation and an increased susceptibility to the development of various psychiatric and neurological disorders, including autism and other neurodevelopmental disorders (NDDs), in the offspring. In the present work, we aimed to provide extensive characterization of the short- and long-term consequences of MIA in the offspring, both at the behavioral and immunological level. To this end, we exposed Wistar rat dams to Lipopolysaccharide and tested the infant, adolescent and adult offspring across several behavioral domains relevant to human psychopathological traits. Furthermore, we also measured plasmatic inflammatory markers both at adolescence and adulthood. Our results support the hypothesis of a deleterious impact of MIA on the neurobehavioral development of the offspring: we found deficits in the communicative, social and cognitive domains, together with stereotypic-like behaviors and an altered inflammatory profile at the systemic level. Although the precise mechanisms underlying the role of neuroinflammatory states in neurodevelopment need to be clarified, this study contributes to a better understanding of the impact of MIA on the risk of developing behavioral deficits and psychiatric illness in the offspring.

Psilocybin mitigates the cognitive deficits observed in a rat model of Fragile X syndrome.

RATIONALE: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability (ID) and the leading monogenic cause of autism spectrum disorder (ASD). Serotonergic neurotransmission has a key role in the modulation of neuronal activity during development, and therefore, it has been hypothesized to be involved in ASD and co-occurring conditions including FXS. As serotonin is involved in synaptic remodeling and maturation, serotonergic insufficiency during childhood may have a compounding effect on brain patterning in neurodevelopmental disorders, manifesting as behavioral and emotional symptoms. Thus, compounds that stimulate serotonergic signaling such as psilocybin may offer promise as effective early interventions for developmental disorders such as ASD and FXS. OBJECTIVES: The aim of the present study was to test whether different protocols of psilocybin administration mitigate cognitive deficits displayed by the recently validated Fmr1-Δexon 8 rat model of ASD, which is also a model of FXS. RESULTS: Our results revealed that systemic and oral administration of psilocybin microdoses normalizes the aberrant cognitive performance displayed by adolescent Fmr1-Δexon 8 rats in the short-term version of the novel object recognition test-a measure of exploratory behavior, perception, and recognition. CONCLUSIONS: These data support the hypothesis that serotonin-modulating drugs such as psilocybin may be useful to ameliorate ASD-related cognitive deficits. Overall, this study provides evidence of the beneficial effects of different schedules of psilocybin treatment in mitigating the short-term cognitive deficit observed in a rat model of FXS.

Anandamide and 2-arachidonoylglycerol differentially modulate autistic-like traits in a genetic model of autism based on FMR1 deletion in rats.

Autism spectrum disorder (ASD) has a multifactorial etiology. Major efforts are underway to understand the neurobiological bases of ASD and to develop efficacious treatment strategies. Recently, the use of cannabinoid compounds in children with neurodevelopmental disorders including ASD has received increasing attention. Beyond anecdotal reports of efficacy, however, there is limited current evidence supporting such an intervention and the clinical studies currently available have intrinsic limitations that make the interpretation of the findings challenging. Furthermore, as the mechanisms underlying the beneficial effects of cannabinoid compounds in neurodevelopmental disorders are still largely unknown, the use of drugs targeting the endocannabinoid system remains controversial. Here, we studied the role of endocannabinoid neurotransmission in the autistic-like traits displayed by the recently validated Fmr1-Δexon 8 rat model of autism. Fmr1-Δexon 8 rats showed reduced anandamide levels in the hippocampus and increased 2-arachidonoylglycerol (2-AG) content in the amygdala. Systemic and intra-hippocampal potentiation of anandamide tone through administration of the anandamide hydrolysis inhibitor URB597 ameliorated the cognitive deficits displayed by Fmr1-Δexon 8 rats along development, as assessed through the novel object and social discrimination tasks. Moreover, blockade of amygdalar 2-AG signaling through intra-amygdala administration of the CB1 receptor antagonist SR141716A prevented the altered sociability displayed by Fmr1-Δexon 8 rats. These findings demonstrate that anandamide and 2-AG differentially modulate specific autistic-like traits in Fmr1-Δexon 8 rats in a brain region-specific manner, suggesting that fine changes in endocannabinoid mechanisms contribute to ASD-related behavioral phenotypes.

Outcomes of early social experiences on glucocorticoid and endocannabinoid systems in the prefrontal cortex of male and female adolescent rats.

Social and emotional experiences differently shape individual's neurodevelopment inducing substantial changes in neurobiological substrates and behavior, particularly when they occur early in life. In this scenario, the present study was aimed at (i) investigating the impact of early social environments on emotional reactivity of adolescent male and female rats and (ii) uncovering the underlying molecular features, focusing on the cortical endocannabinoid (eCB) and glucocorticoid systems. To this aim, we applied a protocol of environmental manipulation based on early postnatal socially enriched or impoverished conditions. Social enrichment was realized through communal nesting (CN). Conversely, an early social isolation (ESI) protocol was applied (post-natal days 14-21) to mimic an adverse early social environment. The two forms of social manipulation resulted in specific behavioral and molecular outcomes in both male and female rat offspring. Despite the combination of CN and ESI did not affect emotional reactivity in both sexes, the molecular results reveal that the preventive exposure to CN differently altered mRNA and protein expression of the main components of the glucocorticoid and eCB systems in male and female rats. In particular, adolescent females exposed to the combination of CN and ESI showed increased corticosterone levels, unaltered genomic glucocorticoid receptor, reduced cannabinoid receptor type-1 and fatty acid amide hydrolase protein levels, suggesting that the CN condition evokes different reorganization of these systems in males and females.

FMR1 deletion in rats induces hyperactivity with no changes in striatal dopamine transporter availability.

Autism Spectrum Disorder (ASD) is a pervasive neurodevelopmental disorder emerging in early life characterized by impairments in social interaction, poor verbal and non-verbal communication, and repetitive patterns of behaviors. Among the best-known genetic risk factors for ASD, there are mutations causing the loss of the Fragile X Messenger Ribonucleoprotein 1 (FMRP) leading to Fragile X syndrome (FXS), a common form of inherited intellectual disability and the leading monogenic cause of ASD. Being a pivotal regulator of motor activity, motivation, attention, and reward processing, dopaminergic neurotransmission has a key role in several neuropsychiatric disorders, including ASD. Fmr1 Δexon 8 rats have been validated as a genetic model of ASD based on FMR1 deletion, and they are also a rat model of FXS. Here, we performed behavioral, biochemical and in vivo SPECT neuroimaging experiments to investigate whether Fmr1 Δexon 8 rats display ASD-like repetitive behaviors associated with changes in striatal dopamine transporter (DAT) availability assessed through in vivo SPECT neuroimaging. At the behavioral level, Fmr1 Δexon 8 rats displayed hyperactivity in the open field test in the absence of repetitive behaviors in the hole board test. However, these behavioral alterations were not associated with changes in striatal DAT availability as assessed by non-invasive in vivo SPECT and Western blot analyses.

Regulation of oxytocin receptor gene expression in obsessive-compulsive disorder: a possible role for the microbiota-host epigenetic axis.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a prevalent and severe clinical condition. Robust evidence suggests a gene-environment interplay in its etiopathogenesis, yet the underlying molecular clues remain only partially understood. In order to further deepen our understanding of OCD, it is essential to ascertain how genes interact with environmental risk factors, a cross-talk that is thought to be mediated by epigenetic mechanisms. The human microbiota may be a key player, because bacterial metabolites can act as epigenetic modulators. We analyzed, in the blood and saliva of OCD subjects and healthy controls, the transcriptional regulation of the oxytocin receptor gene and, in saliva, also the different levels of major phyla. We also investigated the same molecular mechanisms in specific brain regions of socially isolated rats showing stereotyped behaviors reminiscent of OCD as well as short chain fatty acid levels in the feces of rats. RESULTS: Higher levels of oxytocin receptor gene DNA methylation, inversely correlated with gene expression, were observed in the blood as well as saliva of OCD subjects when compared to controls. Moreover, Actinobacteria also resulted higher in OCD and directly correlated with oxytocin receptor gene epigenetic alterations. The same pattern of changes was present in the prefrontal cortex of socially-isolated rats, where also altered levels of fecal butyrate were observed at the beginning of the isolation procedure. CONCLUSIONS: This is the first demonstration of an interplay between microbiota modulation and epigenetic regulation of gene expression in OCD, opening new avenues for the understanding of disease trajectories and for the development of new therapeutic strategies.

Brain Cholesterol Biosynthetic Pathway Is Altered in a Preclinical Model of Fragile X Syndrome.

Fragile X Syndrome (FXS) is the most frequent form of inherited X-linked pathology, associated with an intellectual and developmental disability, and currently considered the first monogenic cause of autism spectrum disorder (ASD). Low levels of total cholesterol reported in the serum of FXS patients, and evidence that FMRP targets a subset of mRNAs encoding proteins of lipid synthesis and transport suggests that the cholesterol metabolism impairments could be involved in FXS. Thus, the aim of the presented work was to investigate the modulations of the cholesterol biosynthetic pathway and its end-products in a recently developed Fmr1-Δexon 8 rat model of FXS. Here, we show that this experimental model mimics what is found in FXS patients, exhibiting a lower serum cholesterol content, accompanied by a reduction in food intake and body weight compared to WT animals. Moreover, alterations of proteins committed to cholesterol synthesis and uptake have been observed in the amygdala, prefrontal cortex and nucleus accumbens. Interestingly, the end-products show a brain region-dependent modulation in Fmr1-Δexon 8 rats. Overall, our results demonstrate that the cholesterol biosynthetic pathway is altered in some brain regions of this preclinical model of FXS. This finding has relevance for future studies to delve deeper into the involvement of this metabolic process in FXS, and thus its possible role as a therapeutic target.

Perinatal supplementation with omega-3 fatty acids corrects the aberrant social and cognitive traits observed in a genetic model of autism based on FMR1 deletion in rats.

Background and objective: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder for which no treatments exist. Fragile X syndrome (FXS) is the most common form of inherited mental retardation and the most frequent monogenic cause of ASD. Given the lack of pharmacological treatments for ASD, increasing interest is devoted to non-pharmacological approaches, including dietary interventions. Omega-3 polyunsaturated fatty acids (PUFAs) are critical for neurobehavioraldevelopment. This study had two aims: 1. To validatethe recently developed Fmr1-Δexon 8 rat model of FXS; 2. To assess the impact of omega-3 PUFAs dietary supplementation during pregnancy and lactation on the altered behavior displayed by Fmr1-Δexon 8 rats.Methods: Female Fmr1-Δexon 8 and wild-type Sprague-Dawley rats were fed with either an omega-3 PUFAs enriched diet or with an isocaloric control diet during pregnancy and lactation. Behavioral experiments were carried out on the infant (Postnatal days (PNDs) 9 and 13), juvenile (PND 35) and adult (PND 90) male offspring.Results: Fmr1-Δexon 8 pups showed hypolocomotion, reduced ultrasonic vocalizations (USVs) emission and impaired social discrimination compared to wild-type controls. Juvenile and adult Fmr1-Δexon 8 rats showed deficits in the social and cognitive domains, that were counteracted by perinatal omega-3 PUFAs supplementation.Conclusion: Our results support the validity of the Fmr1-Δexon 8 rat model to mimic key autistic-like features and support an important role of omega-3 PUFAs during of neurodevelopment. Although the mechanisms underlying the beneficial effects of omega-3 PUFAs supplementation in ASD needs to be clarified, this dietary intervention holds promise to mitigate core and comorbid autistic features.

Missense mutation of Fmr1 results in impaired AMPAR-mediated plasticity and socio-cognitive deficits in mice.

Fragile X syndrome (FXS) is the most frequent form of inherited intellectual disability and the best-described monogenic cause of autism. CGG-repeat expansion in the FMR1 gene leads to FMR1 silencing, loss-of-expression of the Fragile X Mental Retardation Protein (FMRP), and is a common cause of FXS. Missense mutations in the FMR1 gene were also identified in FXS patients, including the recurrent FMRP-R138Q mutation. To investigate the mechanisms underlying FXS caused by this mutation, we generated a knock-in mouse model (Fmr1R138Q) expressing the FMRP-R138Q protein. We demonstrate that, in the hippocampus of the Fmr1R138Q mice, neurons show an increased spine density associated with synaptic ultrastructural defects and increased AMPA receptor-surface expression. Combining biochemical assays, high-resolution imaging, electrophysiological recordings, and behavioural testing, we also show that the R138Q mutation results in impaired hippocampal long-term potentiation and socio-cognitive deficits in mice. These findings reveal the functional impact of the FMRP-R138Q mutation in a mouse model of FXS.

The neurochemistry of social reward during development: What have we learned from rodent models?

Social rewards are fundamental to survival and overall health. Several studies suggest that adequate social stimuli during early life are critical for developing appropriate socioemotional and cognitive skills, whereas adverse social experiences negatively affect the proper development of brain and behavior, by increasing the susceptibility to develop neuropsychiatric conditions. Therefore, a better understanding of the neural mechanisms underlying social interactions, and their rewarding components in particular, is an important challenge of current neuroscience research. In this context, preclinical research has a crucial role: Animal models allow to investigate the neurobiological aspects of social reward in order to shed light on possible neurochemical alterations causing aberrant social reward processing in neuropsychiatric diseases, and they allow to test the validity and safety of innovative therapeutic strategies. Here, we discuss preclinical research that has investigated the rewarding properties of two forms of social interaction that occur in different phases of the lifespan of mammals, that is, mother-infant interaction and social interactions with peers, by focusing on the main neurotransmitter systems mediating their rewarding components. Together, the research performed so far helped to elucidate the mechanisms of social reward and its psychobiological components throughout development, thus increasing our understanding of the neurobiological substrates sustaining social functioning in health conditions and social dysfunction in major psychiatric disorders.

Binding of direct oral anticoagulants to the FA1 site of human serum albumin.

The anticoagulant therapy is widely used to prevent and treat thromboembolic events. Until the last decade, vitamin K antagonists were the only available oral anticoagulants; recently, direct oral anticoagulants (DOACs) have been developed. Since 55% to 95% of DOACs are bound to plasma proteins, the in silico docking and ligand-binding properties of drugs apixaban, betrixaban, dabigatran, edoxaban, and rivaroxaban and of the prodrug dabigatran etexilate to human serum albumin (HSA), the most abundant plasma protein, have been investigated. DOACs bind to the fatty acid (FA) site 1 (FA1) of ligand-free HSA, whereas they bind to the FA8 and FA9 sites of heme-Fe(III)- and myristic acid-bound HSA. DOACs binding to the FA1 site of ligand-free HSA has been validated by competitive inhibition of heme-Fe(III) recognition. Values of the dissociation equilibrium constant for DOACs binding to the FA1 site (ie, calc KDOAC ) derived from in silico docking simulations (ranging between 1.2 × 10-8 M and 1.4 × 10-6 M) agree with those determined experimentally from competitive inhibition of heme-Fe(III) binding (ie, exp KDOAC ; ranging between 2.5 × 10-7 M and 2.2 × 10-6 M). In addition, this study highlights the inequivalence of rivaroxaban binding to mammalian serum albumin. Given the HSA concentration in vivo (~7.5 × 10-4 M), values of KDOAC here determined indicate that the formation of the HSA:DOACs complexes in the absence and presence of FAs and heme-Fe(III) may occur in vivo. Therefore, HSA appears to be an important determinant for DOACs transport.

Detrimental effects of the 'bath salt' methylenedioxypyrovalerone on social play behavior in male rats.

Methylenedioxypyrovalerone (MDPV) is the most popular synthetic cathinone found in products marketed as 'bath salts', widely abused among teenagers and young adults. Synthetic cathinones have pharmacological effects resembling those of psychostimulants, which are known to disrupt a variety of social behaviors. However, despite the popular use of MDPV by young people in social contexts, information about its effects on social behavior is scarce. To investigate the impact of MDPV on social behavior at young age, and the underlying neurobehavioral mechanisms, we focused on social play behavior. Social play behavior is the most characteristic social behavior displayed by young mammals and it is crucial for neurobehavioral development. Treatment with MDPV reduced social play behavior in both juvenile and young adult male rats, and its play-suppressant effect was subject to tolerance but not sensitization. As the behavioral effects of MDPV have been ascribed to dopaminergic and noradrenergic neurotransmission, and given the role of these neurotransmitters in social play, we investigated the involvement of dopamine and noradrenaline in the play-suppressant effects of MDPV. The effects of MDPV on social play were blocked by either the α2 adrenoceptor antagonist RX821002 or the dopamine receptor antagonist flupenthixol, given alone or together at sub-effective doses. In sum, MDPV selectively suppresses the most vigorous social behavior of developing rats through both noradrenergic and dopaminergic mechanisms. This study provides important preclinical evidence of the deleterious effects of MDPV on social behavior, and as such increases our understanding of the neurobehavioral effects of this popular cathinone.