A systematic review of preclinical studies exploring the role of insulin signalling in executive function and memory.

Beside its involvement in somatic dysfunctions, altered insulin signalling constitutes a risk factor for the development of mental disorders like Alzheimer's disease and obsessive-compulsive disorder. While insulin-related somatic and mental disorders are often comorbid, the fundamental mechanisms underlying this association are still elusive. Studies conducted in rodent models appear well suited to help decipher these mechanisms. Specifically, these models are apt to prospective studies in which causative mechanisms can be manipulated via multiple tools (e.g., genetically engineered models and environmental interventions), and experimentally dissociated to control for potential confounding factors. Here, we provide a narrative synthesis of preclinical studies investigating the association between hyperglycaemia - as a proxy of insulin-related metabolic dysfunctions - and impairments in working and spatial memory, and attention. Ultimately, this review will advance our knowledge on the role of glucose metabolism in the comorbidity between somatic and mental illnesses.

Methylation Dynamics on 5'-UTR of DAT1 Gene as a Bio-Marker to Recognize Therapy Success in ADHD Children.

Attention-deficit/hyperactivity disorder (ADHD), a neuropsychiatric condition characterized by inattention, hyperactivity, and impulsivity, afflicts 5% of children worldwide. Each ADHD patient presents with individual cognitive and motivational peculiarities. Furthermore, choice of appropriate therapy is still up to clinicians, who express somewhat qualitative advice on whether a child is being successfully cured or not: it would be more appropriate to use an objective biomarker to indicate whether a treatment led to benefits or not. The aim of our work is to search for such clinical biomarkers. We recruited 60 ADHD kids; psychopathological scales were administered at recruitment and after six weeks of therapy. Out of such a cohort of ADHD children, we rigorously extracted two specific subgroups; regardless of the initial severity of their disease, we compared those who obtained the largest improvement (ΔCGAS > 5) vs. those who were still characterized by a severe condition (CGAS < 40). After such a therapy, methylation levels of DNA extracted from buccal swabs were measured in the 5'-UTR of the DAT1 gene. CpGs 3 and 5 displayed, in relation to the other CpGs, a particular symmetrical pattern; for "improving" ADHD children, they were methylated together with CpG 2 and CpG 6; instead, for "severe" ADHD children, they accompanied a methylated CpG 1. These specific patterns of methylation could be used as objective molecular biomarkers of successful cures, establishing if a certain therapy is akin to a given patient (personalized medicine). Present data support the use of post-therapy molecular data obtained with non-invasive techniques.

Methylation patterns within 5'-UTR of DAT1 gene as a function of allelic 3'-UTR variants and their maternal or paternal origin: May these affect the psychopathological phenotypes in children? An explorative study.

Psychopathological symptoms such as depression/anxiety vs attention or aggression problems, in children, have been associated to altered expression of the DAT1/SLC6A3 gene. Inheriting specific 9- or 10-repeat VNTR alleles could modify the pattern of methylation in the CpGs islands at the 5'-UTR of the DAT1 gene. Through accurate recruitment at primary schools, we ended up with four subgroups of children: 9/9 and 10/10 homozygous; 9/10 heterozygous born from 9/10 mothers and 10/10 fathers (called heM); 9/10 heterozygous born from 10/10 mothers and 9/10 fathers (called heF). (Epi)genetical changes were found to be in relation to internalizing and externalizing symptoms: compared to other genotypes, our 9/9 children exhibited mainly internalizing symptoms, while 10/10 genotype was previously associated with ADHD severity. We found that 10/10 children bear 5'-UTR motifs showing a CpGs 1-2-3-5 unity with anticorrelated CpG 6, while 9/9 children showed rather a demethylated CpG 1 linked to demethylated CpG 6. We found two different patterns between heMs and heFs: a feature of heM children is in CpGs 1-3 methylated pattern with CpGs 2, 5 and 6 demethylated together, supporting a "split" unitary destiny. Within the heF children, the status for CpGs 3 + 6 remained opposite, yet pattern of (de)methylation was not well defined. The prevailing one between inherited parental alleles may somewhat influence the motif destiny of heterozigous children. Present work aimed to identify novel epigenetic biomarkers, to be exploited as fairly indicators of children's psychopathological vulnerability.

The presence of a potential competitor modulates risk preferences in rats.

Although both human and non-human animals, in everyday life, deal with risky decisions in a social environment, few studies investigated how social dimension influences risk preferences (i.e., if consequences on others feeds back over own choice). Here, we assessed whether the presence of a conspecific, acting as a potential competitor for the same food resource, influenced risky decision-making in male rats. Subjects received a series of choices between a safe option (always yielding a small yet optimal reward, solely to itself) and a risky option (yielding a larger but suboptimal reward, one third of times to itself and two third of times delivered to the other half cage); rats were tested twice, both alone and paired with a conspecific, recipient of own-lost food and hence acting as potential competitor. Results showed that focal subjects were more risk-prone when paired with a conspecific than when tested alone. However, rats exhibited also a higher motivational conflict with a competing bystander present than alone: data suggest that the primary drive was to increase "own" food rather than either a competitive or prosocial tendency. Overall, for rats tested in a risky-choice task, a competitive social context increased the salience and attractiveness of larger food outcomes, as observed in humans and great apes. This led to the economically irrational response of selecting the "binge-but-risky" option, notwithstanding uncertainty about the actual recipient of such food.

Truncated dopamine transporter's epigenetics: Heterozigosity of the grandmother rat temperates the vulnerable phenotype in second-generation offspring.

Behavioral phenotype differs among epigenotypes of dopamine-transporter heterozygous (DAT-HET) rats. Epigenetic regulations act through transgenerational effects, referring to phenotypic variations emerging at second or third generation. To investigate transgenerational influences exerted by maternal grandmothers, we developed breeding schemes where only the genotype of maternal grandmothers varied. HET females, to serve as MAT vs. MIX mothers, were generated, respectively, from WT × KO = MAT and MAT × KO = MIX breeding, with KO males acting as grandfather. The HET experimental groups, generated from either MAT or MIX mothers, were called MIX-by-MAT and MIX2 (male-fathers KO; asset-M: wild/healthy-allele from dam) or SOT and SIX (male-fathers WT; asset-P: mutated-allele from dam). Thus, sequelae of first encounter between wild/healthy and mutated DAT alleles (in maternal-lineage) were compared at first- (MAT-dam, WT-grandmother) vs. at second- (MIX-dam, HET-grandmother) generation. We characterized, within these epigenotypes, (1) circadian home-cage activity and (2) preference for social stimuli. Marked alterations of circadian activity appeared in MIX-by-MAT HETs, offspring of MAT-dams, compared with MIX2 HET (offspring of MIX-dams); The latter, in turn, were undistinguishable from WT-controls. A clear-cut social preference by WT rats was expressed towards SIX compared with SOT stimulus rats, confirming that the latter elicited reduced social motivations. In conclusion, significant epigenetic modulations took place in DAT-HET rats, as a function of maternal grandmother's genotype.

Social Interactions of Dat-Het Epi-Genotypes Differing for Maternal Origins: The Development of a New Preclinical Model of Socio-Sexual Apathy.

Social interaction is essential for life but is impaired in many psychiatric disorders. We presently focus on rats with a truncated allele for dopamine transporter (DAT). Since heterozygous individuals possess only one non-mutant allele, epigenetic interactions may unmask latent genetic predispositions. Homogeneous "maternal" heterozygous offspring (termed MAT-HET) were born from dopamine-transporter knocked-out (DAT-KO) male rats and wild-type (WT) mothers; "mixed" heterozygous offspring (termed MIX-HET) were born from both DAT-heterozygous parents. Their social behavior was assessed by: partner-preference (PPT), social-preference (SPT) and elicited-preference (EPT) tests. During the PPT, focal MIX-HET and MAT-HET males had a choice between two WT females, one in estrous and the other not. In the SPT, they met as stimulus either a MIX-HET or a WT male. In the EPT, the preference of focal male WT rats towards either a MIX- or a MAT-HET stimulus was tested. MIX-HET focal males showed an abnormal behavior, seeming not interested in socializing either with a female in estrous or with another male if MIX-HET. Focal MAT-HET males, instead, were very attracted by the female in estrous, but totally ignored the MIX-HET male. We assessed the expression of noradrenaline transporter (NET) in prefrontal cortex, hippocampus and hypothalamus, finding differences between the two offspring. MIX-HETs' hypothalamus and hippocampus showed less NET than MAT-HETs, while the latter, in turn, showed higher NET than WTs. These behavioral differences between heterozygous groups may be attributed to different maternal cares received. Results allow preclinical understanding of epigenetic factors involved in social-behavior abnormalities, typical of many psychiatric disorders.

Treatment with the Bacterial Toxin CNF1 Selectively Rescues Cognitive and Brain Mitochondrial Deficits in a Female Mouse Model of Rett Syndrome Carrying a MeCP2-Null Mutation.

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene and a major cause of intellectual disability in females. No cure exists for RTT. We previously reported that the behavioural phenotype and brain mitochondria dysfunction are widely rescued by a single intracerebroventricular injection of the bacterial toxin CNF1 in a RTT mouse model carrying a truncating mutation of the MeCP2 gene (MeCP2-308 mice). Given the heterogeneity of MECP2 mutations in RTT patients, we tested the CNF1 therapeutic efficacy in a mouse model carrying a null mutation (MeCP2-Bird mice). CNF1 selectively rescued cognitive defects, without improving other RTT-related behavioural alterations, and restored brain mitochondrial respiratory chain complex activity in MeCP2-Bird mice. To shed light on the molecular mechanisms underlying the differential CNF1 effects on the behavioural phenotype, we compared treatment effects on relevant signalling cascades in the brain of the two RTT models. CNF1 provided a significant boost of the mTOR activation in MeCP2-308 hippocampus, which was not observed in the MeCP2-Bird model, possibly explaining the differential effects of CNF1. These results demonstrate that CNF1 efficacy depends on the mutation beared by MeCP2-mutated mice, stressing the need of testing potential therapeutic approaches across RTT models.

Callous unemotional trait-like mice and their stressed dams.

The co-occurrence of excess rates of aggression, general violation of societal norms and callous-unemotional trait confers specific risk for adult psychopathy. With the aim to address experimentally a model of conduct disorder, we investigated the male offspring of individual mouse dams characterized by high basal plasma corticosterone concentration (HC trait). Notably, classification indices correlated selectively in these females with quite poor maternal care devoted to their offspring. Contrary to their HC mothers, adult male offspring exhibited an integrated profile of dampened physiological reactivity to external stressors co-occurring poor sociability/emotional contagion, impaired punishment-induced memory, and exacerbated aggression. A significant reduction of glucocorticoid and opioid mu receptors' expression in frontal cortex of model HC offspring was also evidenced. Moreover, in the absence of changes in oxytocin receptor in behaviorally-relevant neural areas, we showed that intranasal oxytocin administration (0 or 20.0 µg/kg) selectively modulated specific components of the behavioral phenotype. Ultimately, current data support the notion that maternally-inoculated environmental stress early in development may represent a critical risk factor in disturbances characterised by abnormal aggression and excess callousness.

Aberrant Early in Life Stimulation of the Stress-Response System Affects Emotional Contagion and Oxytocin Regulation in Adult Male Mice.

Results over the last decades have provided evidence suggesting that HPA axis dysfunction is a major risk factor predisposing to the development of psychopathological behaviour. This susceptibility can be programmed during developmental windows of marked neuroplasticity, allowing early-life adversity to convey vulnerability to mental illness later in life. Besides genetic predisposition, also environmental factors play a pivotal role in this process, through embodiment of the mother's emotions, or via nutrients and hormones transferred through the placenta and the maternal milk. The aim of the current translational study was to mimic a severe stress condition by exposing female CD-1 mouse dams to abnormal levels of corticosterone (80 µg/mL) in the drinking water either during the last week of pregnancy (PreCORT) or the first one of lactation (PostCORT), compared to an Animal Facility Rearing (AFR) control group. When tested as adults, male mice from PostCORT offspring and somewhat less the PreCORT mice exhibited a markedly increased corticosterone response to acute restraint stress, compared to perinatal AFR controls. Aberrant persistence of adolescence-typical increased interest towards novel social stimuli and somewhat deficient emotional contagion also characterised profiles in both perinatal-CORT groups. Intranasal oxytocin (0 or 20.0 µg/kg) generally managed to reduce the stress response and restore a regular behavioural phenotype. Alterations in density of glucocorticoid and mineralocorticoid receptors, oxytocin and µ- and κ-opioid receptors were found. Changes differed as a function of brain areas and the specific age window of perinatal aberrant stimulation of the HPA axis. Present results provided experimental evidence in a translational mouse model that precocious adversity represents a risk factor predisposing to the development of psychopathological behaviour.

"Himalayan Bridge": A New Unstable Suspended Bridge to Investigate Rodents' Venturesome Behavior.

While both risk-taking and avoidant behaviors are necessary for survival, their imbalanced expression can lead to impulse-control and anxiety disorders, respectively. In laboratory rodents, the conflict between risk proneness and anxiety can be studied by using their innate fear of heights. To explore this aspect in detail and investigate venturesome behavior, here we used a "Himalayan Bridge," a rat-adapted version of the suspended wire bridge protocol originally developed for mice. The apparatus is composed of two elevated scaffolds connected by bridges of different lengths and stability at 1 m above a foam rubber-covered floor. Rats were allowed to cross the bridge to reach food, and crossings, pawslips, turnabouts, and latencies to cross were measured. Given the link between risky behavior and adolescence, we used this apparatus to investigate the different responses elicited by a homecage mate on the adolescent development of risk-taking behavior. Thus, 24 wild-type (WT) subjects were divided into three different housing groups: WT rats grown up with WT adult rats; control WT adolescent rats (grown up with WT adolescents), which showed a proclivity to risk; and WT rats grown up with an adult rat harboring a truncated mutation for their dopamine transporter (DAT). This latter group exhibited risk-averse responses reminiscent of lower venturesomeness. Our results suggest that the Himalayan Bridge may be useful to investigate risk perception and seeking; thus, it should be included in the behavioral phenotyping of rat models of psychiatric disorders and cognitive dysfunctions.

Stimulation of the Serotonin Receptor 7 Restores Brain Histone H3 Acetylation and MeCP2 Corepressor Protein Levels in a Female Mouse Model of Rett Syndrome.

Rett syndrome (RTT) is a rare neurological disorder caused by mutations in the X-linked MECP2 gene, characterized by severe behavioral and physiological impairments for which no cure is available. The stimulation of serotonin receptor 7 (5-HT7R) with its selective agonist LP-211 (0.25 mg/kg/day for 7 days) was proved to rescue neurobehavioral alterations in a mouse model of RTT. In the present study, we aimed at gaining insight into the mechanisms underpinning the efficacy of 5-HT7R pharmacological stimulation by investigating its epigenetic outcomes in the brain of RTT female mice bearing a truncating MeCP2 mutation. Treatment with LP-211 normalized the reduced histone H3 acetylation and HDAC3/NCoR levels, and increased HDAC1/Sin3a expression in RTT mouse cortex. Repeated 5-HT7R stimulation also appeared to strengthen the association between NCoR and MeCP2 in the same brain region. A different profile was found in RTT hippocampus, where LP-211 rescued H3 hyperacetylation and increased HDAC3 levels. Overall, the present data highlight a new scenario on the relationship between histone acetylation and serotoninergic pathways. 5-HT7R is confirmed as a pivotal therapeutic target for the recovery of neuronal function supporting the translational value of this promising pharmacological approach for RTT.

Publisher Correction: Mice repeatedly exposed to Group-A ß-Haemolytic Streptococcus show perseverative behaviors, impaired sensorimotor gating, and immune activation in rostral diencephalon.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Striatal dynamics as determinants of reduced gambling vulnerability in the NHE rat model of ADHD.

The Naples High-Excitability (NHE) is a validated rat strain to model for a mesocortical variant of Attention Deficit Hyperactivity Disorder (ADHD). NHE rats' brains have a tuned-down cortical and a potentiated limbic loop (Zoratto et al., 2017). ADHD and comorbid pathological gambling (PG) involve similar deficits of prefrontal-striatal dialogue. This work aimed to understand if NHE rats (compared to normal random-bred rats, NRB) can be a useful model for gambling vulnerability in ADHD. Experiment 1 evaluated gambling proneness in NHE rats, namely attraction/avoidance in nose-poking for a "Large & Luck-Linked" (LLL) reward (versus a "Small & Sure" one, SS), when the probability of LLL delivery was progressively reduced. Experiment 2 assessed (by phMRI) differential responsivity of ventral (vStr) versus dorsal (dStr) striatum following a methylphenidate (MPH, 4 mg/kg I.P.) challenge. In NHE rats, reduced attraction by secondary cues (associated with uncertain, rarefying LLL delivery) comes along with little or no activation of dStr and enhanced activation of vStr by MPH. Together, such evidences from NHE rats indicate distinctive roles of ventral (enhanced value given to actual primary reward) and dorsal (lower encoding of repeated stimulus-reward associations into a habit) striatum. In conclusion, the dynamics of reward systems could link an attention deficit with a decreased vulnerability to pathological gambling.

Anatomical and behavioral impact of a lentiviral tool tapping onto hippocampal serotonin reuptake in rats.

We aimed at the further characterization of rats in which SERT gene silencing was achieved by hippocampal injection of a lentiviral vector, carrying three si-RNA to block SERT mRNA at 66% of normal levels. Improved self-control and reduced restlessness were already demonstrated in these rats. Present further studies consisted of male adult rats, bilaterally inoculated within the hippocampus; control rats received lentivirus particles inactivated with heat. Both groups were maintained in isolation for 5 months, starting from inoculation. Neurochemical changes were studied by proton magnetic resonance spectroscopy (1H-MRS): we found increased hippocampal viability and bioenergetic potential; however, rats showed a behaviorally depressive pattern, also characterized by enhanced affiliation. Based on the extent of such effects, the whole lenti-SERT group was divided into two subgroups, termed intermediate- and extreme- phenotype profiles. While all rats had a widespread modification within dorsal/ventral striatum, amygdala, and hypothalamus, only the former subgroup showed an involvement of Raphé medialis, while, for the latter subgroup, an increase of SERT within hippocampus was unexpectedly caused. Within the less-affected "intermediate" rats, hippocampal 5-HT7 receptors were down-modulated, and also similarly within substantia nigra, septum, and neocortex. This picture demonstrates that additional rather than fewer neurobiological changes accompany a lower phenotypic expression. Overall, tapping hippocampal SERT affected the balance between habits versus strategies of coping by promoting morphogenetic processes indicative of a serotonergic fiber plasticity. Supplementary studies about serotonergic dynamics and neurogenesis within fronto-striatal circuits are needed.

Brain-Immune Alterations and Mitochondrial Dysfunctions in a Mouse Model of Paediatric Autoimmune Disorder Associated with Streptococcus: Exacerbation by Chronic Psychosocial Stress.

Adverse psychosocial experiences have been shown to modulate individual responses to immune challenges and affect mitochondrial functions. The aim of this study was to investigate inflammation and immune responses as well as mitochondrial bioenergetics in an experimental model of Paediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus (PANDAS). Starting in adolescence (postnatal day 28), male SJL/J mice were exposed to five injections (interspaced by two weeks) with Group-A beta-haemolytic streptococcus (GAS) homogenate. Mice were exposed to chronic psychosocial stress, in the form of protracted visual exposure to an aggressive conspecific, for four weeks. Our results indicate that psychosocial stress exacerbated individual response to GAS administrations whereby mice exposed to both treatments exhibited altered cytokine and immune-related enzyme expression in the hippocampus and hypothalamus. Additionally, they showed impaired mitochondrial respiratory chain complexes IV and V, and reduced adenosine triphosphate (ATP) production by mitochondria and ATP content. These brain abnormalities, observed in GAS-Stress mice, were associated with blunted titers of plasma corticosterone. Present data support the hypothesis that challenging environmental conditions, in terms of chronic psychosocial stress, may exacerbate the long-term consequences of exposure to GAS processes through the promotion of central immunomodulatory and oxidative stress.

Reduced adolescent risk-assessment and lower nicotinic beta-2 expression in rats exposed to nicotine through lactation by forcedly drinking dams.

Few animal studies focus on consequences of nicotine postnatal exposure, particularly through lactation. We have recently shown that forced nicotine drinking elevates maternal care, paradoxically provoking arousal and stress in pups. Present work aimed to evaluate the specific contribution of altered maternal cares, compared to the sequelae merely due to nicotine effects. Two groups were compared to water-drinking control dams: (i) free-choice dams (H2O+NIC group) drinking from two bottles, containing either nicotine or water; (ii) forced dams (NIC+NIC group) drinking from two bottles, both containing nicotine. We previously demonstrated that nicotine was indeed transferred to the lactating offspring. Regarding behavioural consequences at adolescence, both H2O+NIC and NIC+NIC rats were slower than controls in discovering a novel over a familiar compartment, whilst only NIC+NIC rats exhibited reduced risk-related avoidance and assessment behaviour. Brain analyses at adulthood suggest that, in prefrontal cortex, nicotine per se reduced serotonin, while the maternal overcare reduced CHRN-B2 gene-expression. As a whole, unescapable nicotine-enhanced maternal care could have an impact on the offspring arousal by acting on prefrontal CHRN-B2 gene-expression. When present results are translated to consequences of non-voluntary exposure in humans, we propose that children receiving altered attentions by a smoking caregiver might undergo a neuro-behavioural development biased towards emotional shyness.

Rett syndrome before regression: A time window of overlooked opportunities for diagnosis and intervention.

Rett syndrome (RTT) is a rare neurological disorder primarily affecting females, causing severe cognitive, social, motor and physiological impairments for which no cure currently exists. RTT clinical diagnosis is based on the peculiar progression of the disease, since patients show an apparently normal initial development with a subsequent sudden regression at around 2 years of age. Accumulating evidences are rising doubts regarding the absence of early impairments, hence questioning the concept of regression. We reviewed the published literature addressing the pre-symptomatic stage of the disease in both patients and animal models with a particular focus on behavioral, physiological and brain abnormalities. The emerging picture delineates subtle, but reliable impairments that precede the onset of overt symptoms whose bases are likely set up already during embryogenesis. Some of the outlined alterations appear transient, suggesting compensatory mechanisms to occur in the course of development. There is urgent need for more systematic developmental analyses able to detect early pathological markers to be used as diagnostic tools and precocious targets of time-specific interventions.