Exploring transgenerational inheritance in epigenotypes of DAT heterozygous rats: Circadian anomalies and attentional vulnerability.

Dopamine (DA) is mainly involved in locomotor activity, reward processes and maternal behaviors. Rats with KO gene for dopamine transporter (DAT), coding for a truncated DAT protein, are in hyperdopaminergic conditions and thus develop stereotyped behaviors and hyperactivity. Our aim was to test the prior transgenerational modulation of wild and truncated alleles as expressed in heterozygous DAT rats: specifically, we addressed the possible sequelae due to genotype and gender of the ancestors, with regard to behavioral differences in F1, F2, F3 rats. We studied non-classical DAT heterozygotes (HETs) based on two specular lines, with putative grand-maternal vs. grand-paternal imprinting. MAT females (F1; offspring of KO male and WT female) mated with a KO male to generate MIX offspring (F2). Specularly, PAT females (F1; offspring of KO female and WT male) mated with a KO male to generate PIX offspring (F2). Similarly to PAT, we obtained MUX (F2; HET offspring of MAT sire and KO dam); we also observed the F3 (MYX: HET offspring of KO male and MUX female, thus with DAT-KO maternal grandmother like also for PIX). We studied their circadian cycle of locomotor activity and their behavior in the elevated-plus-maze (EPM). Locomotor hyper-activity occurs in F1, the opposite occurs in F2, with MYX rats appearing undistinguishable from WT ones. Open-arm preference emerged in PIX and MIX rats. Only MAT and MYX rats showed a significant vulnerability for ADHD-like inattentive symptoms (duration of rearing in the EPM; Viggiano et al., 2002). A risk-taking profile is evident in the F2 phenotype, while inattentiveness from F1 progeny tends to be transferred to F3. We hypothesize that DAT-related phenotypes result from effective inheritance through pedigree of imprints that are dependent on grandparents, suggesting a protective role for gestation within a hyperdopaminergic uterus. For major features, similar odd (F1, F3) generations appear opposed to even (F2) ones; for minor specific features, the phenotype transfer may affect the progenies with a male but not a female DAT-KO ancestor.

Selective alterations of endocannabinoid system genes expression in obsessive compulsive disorder.

Obsessive Compulsive Disorder (OCD) is listed as one of the top 10 most disabling neuropsychiatric conditions in the world. The neurobiology of OCD has not been completely understood and efforts are needed in order to develop new treatments. Beside the classical neurotransmitter systems and signalling pathways implicated in OCD, the possible involvement of the endocannabinoid system (ECS) has emerged in pathophysiology of OCD. We report here selective downregulation of the genes coding for enzymes allowing the synthesis of the endocannabinoids. We found reduced DAGLα and NAPE-PLD in blood samples of individuals with OCD (when compared to healthy controls) as well as in the amygdala complex and prefrontal cortex of dopamine transporter (DAT) heterozygous rats, manifesting compulsive behaviours. Also mRNA levels of the genes coding for cannabinoid receptors type 1 and type 2 resulted downregulated, respectively in the rat amygdala and in human blood. Moreover, NAPE-PLD changes in gene expression resulted to be associated with an increase in DNA methylation at gene promoter, and the modulation of this gene in OCD appears to be correlated to the progression of the disease. Finally, the alterations observed in ECS genes expression appears to be correlated with the modulation in oxytocin receptor gene expression, consistently with what recently reported. Overall, we confirm here a role for ECS in OCD at both preclinical and clinical level. Many potential biomarkers are suggested among its components, in particular NAPE-PLD, that might be of help for a prompt and clear diagnosis.

Mild internet use is associated with epigenetic alterations of key neurotransmission genes in salivary DNA of young university students.

The potentially problematic use of the Internet is a growing concern worldwide, which causes and consequences are not completely understood yet. The neurobiology of Internet addiction (IA) has attracted much attention in scientific research, which is now focusing on identifying measurable biological markers. Aim of this study was to investigate epigenetic and genetic regulation of oxytocin receptor (OXTR), dopamine transporter (DAT1) and serotonin transporter (SERT) genes using DNA obtained from saliva samples of young university students: the Internet Addiction Test (IAT) was administered to evaluate the potential existence and intensity of IA. Significant changes in DNA methylation levels at OXTR, DAT1 and SERT genes were observed in the 30 < IAT < 49 group (mild-risk internet users) compared to the IAT < 29 subjects (complete control of internet use) and IAT > 50 subjects (considered as moderately addicted). Moreover, epigenetic markers were significantly correlated, either directly (for OXTR and DAT1) or inversely (OXTR and DAT1 versus SERT), to the psychometric properties. Our data confirmed the association of OXTR, DAT1 and SERT genes in processes related to behavioural addictions and might be of relevance to suggest possible biological predictors of altered behaviours and the eventual vulnerability to develop an IA. Different other genetic pathways have been suggested to play a role in IA and research is ongoing to better define them, in order to help in the early diagnosis as well as in the development of new potential treatments.

DAT1 5'-Un-Translated-Region Methylation Patterns as Bio-Markers of ADHD Psycho-Pathology: Contribution to Disease Prognosis and to Monitoring of a Successful Therapy.

Epigenetic modifications, such as changes in DNA methylation, have been linked to several diseases in recent years. The purpose of our study was to search for biomarkers that (using non-invasive techniques) could assist the clinician in the prognosis of infant/adolescent psychopathology. We previously showed that changes in methylation of the 5'-UTR in the DAT1/SLC6A3 gene can be used as a biomarker for the prognosis of initial severe ADHD: treatment-resistant severe ADHD children were characterized by methylated CpG 1 in particular, while methylated CpGs 2 and 6 were then found in children who improved after the therapy. Further, we confirmed these outcomes and provided the hypothesis that symptomatology might be influenced by the children's genotype and family environment. In particular, levels of CpG 3 methylation in the heterozygous ADHD children were associated with high paternal own risk or stress. Eventually, we found that the same biomarkers are more broadly useful in the field of internalizing or externalizing symptoms (when a certain vulnerability is already present in the child). In particular, it was seen how inheriting specific 9-repeat or 10-repeat VNTR alleles from the mother or from the father could modify the pattern of methylation at the 5'-UTR of the DAT1 gene. A specific pattern of methylations (with CpG 2 following either CpGs 1 + 3 or CpG 6 at the DAT1 5'-UTR) has been associated, therefore, with the likelihood of an internalizing or externalizing developmental trajectory entailing ADHD-like psycho-pathological characteristics. Since each individual responds differently to a specific treatment, we suggest that these methylation patterns may be used as biomarkers to monitor the outcome and/or predict the success of a given therapy (personalized medicine).

Characterization of Behavioral Phenotypes in Heterozygous DAT Rat Based on Pedigree.

Dopamine is an essential neurotransmitter whose key roles include movement control, pleasure and reward, attentional and cognitive skills, and regulation of the sleep/wake cycle. Reuptake is carried out by the dopamine transporter (DAT; DAT1 SLC6A3 gene). In order to study the effects of hyper-dopaminergia syndrome, the gene was silenced in rats. DAT-KO rats show stereotypical behavior, hyperactivity, a deficit in working memory, and an altered circadian cycle. In addition to KO rats, heterozygous (DAT-HET) rats show relative hypofunction of DAT; exact phenotypic effects are still unknown and may depend on whether the sire or the dam was KO. Our goal was to elucidate the potential importance of the parental origin of the healthy or silenced allele and its impact across generations, along with the potential variations in maternal care. We thus generated specular lines to study the effects of (grand) parental roles in inheriting the wild or mutated allele. MAT-HETs are the progeny of a KO sire and a WT dam; by breeding MAT-HET males and KO females, we obtained subjects with a DAT -/- epigenotype, named QULL, to reflect additional epigenetic DAT modulation when embryos develop within a hyper-dopaminergic KO uterus. We aimed to verify if any behavioral anomaly was introduced by a QULL (instead of KO) rat acting as a direct father or indirect maternal grandfather (or both). We thus followed epigenotypes obtained after three generations and observed actual effects on impaired maternal care of the offspring (based on pedigree). In particular, offspring of MAT-HET-dam × QULL-sire breeding showed a compulsive and obsessive phenotype. Although the experimental groups were all heterozygous, the impact of having a sire of epigenotype QULL (who developed in the uterus of a KO grand-dam) has emerged clearly. Along the generations, the effects of the DAT epigenotype on the obsessive/compulsive phenotype do vary as a function of the uterine impact on either allele in one's genealogical line.

Inheritance of wild and truncated DAT alleles from grand-parents: Opposite transgenerational consequences on the behavioral phenotype in adolescent DAT heterozygous rats.

Dopamine plays important roles in implicit memory and motivation of behavior. Environmental inputs can produce transgenerational epigenetic changes. This concept also includes the uterus: experimentally, we sought to create hyper-dopaminergic uterine conditions through ineffective dopamine-transporter (DAT) protein, obtained by inserting a stop-codon into the SLC6A3 gene. By crossing WT-dam with KO-sire (or vice-versa), we obtained a 100% DAT-heterozygous (HET) offspring with known derivation of the wild allele: MAT rats are offspring of WT-female and KO-male; PAT rats are offspring of KO-female and WT-male. We reconstructed inheritance of alleles, by crossing PAT-male with MAT-female or vice-versa, obtaining GIX (PAT-male with MAT-female) and DIX (MAT-male with PAT-female) rats (such offspring present specular paths in allele inheritance from grandparents). We conducted three experiments: first, we assessed maternal behaviour (four epigenotypes: WT, MAT, PAT and WHZ=HET-pups fostered-to-a WT-dam); in the second, we analysed sleep-wake cycles of GIX and DIX epigenotypes with their WIT siblings as controls; in the third, we explored the impact of WT or MAT mother on WT or HET pups. MAT-dams (with GIX-pups) express excessive licking/grooming. However, in the mere presence of "sick" epigenotype, PAT-dams (with DIX-pups) and also WHZ (i.e., WT-dams but with HET-pups) expressed greater nest-building care towards the offspring, compared to "true-wild" litters (WT-dams with WT-pups). In Exp. 2 at adolescence, GIX epigenotype showed locomotor hyperactivity during late waking-phase, while DIX epigenotype exhibited pronounced hypoactivity compared to controls. In Exp. 3, we confirmed that HET adolescent pups receiving cares from a MAT-dam may develop additional hyperactivity when awake, but additional hypoactivity during rest-hours. Thus, behavioral changes observed in DAT-heterozygous offspring have opposite courses based on of DAT-allele inheritance from a grandparent through the sire or the dam. In conclusion, behavioural changes in the offspring have antithetic courses with respect to inheritance of DAT-allele via sperm or egg.

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.

How Can CpG Methylations, and Pair-to-Pair Correlations between the Main (Gene) and the Opposite Strands, Suggest a Bending DNA Loop: Insights into the 5'-UTR of DAT1.

A working hypothesis issues from patterns of methylation in the 5'-UTR of the DAT1 gene. We considered relationships between pairs of CpGs, of which one on the main-gene strand and another on the complementary opposite strand (COS). We elaborated on data from ADHD children: we calculated all possible combinations of probabilities (estimated by multiplying two raw values of methylation) in pairs of CpGs from either strand. We analyzed all correlations between any given pair and all other pairs. For pairs correlating with M6-M6COS, some pairs had cytosines positioning to the reciprocal right (e.g., M3-M2COS and M6-M5COS), other pairs had cytosines positioning to the reciprocal left (e.g., M2-M3COS; M5-M6COS). Significant pair-to-pair correlations emerged between main-strand and COS CpG pairs. Through graphic representations, we hypothesized that DNA folded to looping conformations: the C1GG C2GG C3GG and C5G C6G motifs would become close enough to allow cytosines 1-2-3 to interact with cytosines 5-6 (on both strands). Data further suggest a sliding, with left- and right-ward oscillations of DNA strands. While thorough empirical verification is needed, we hypothesize simultaneous methylation of main-strand and COS DNA ("methylation dynamics") to serve as a promising biomarker.

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.

Dopamine-transporter heterozygous rats carrying maternal wild-type allele are more vulnerable to the development of compulsive behavior.

Compulsivity is defined as an unstoppable tendency toward repetitive and habitual actions, which are reiterated despite negative consequences. Polydipsia is induced preclinically by intermittent reward, leading rodents to ingest large amounts of fluids. We focused on the role of dopamine transporter (DAT) and inheritance factors in compulsive behavior. Our sample consisted of DAT heterozygous (HET) rats with different genetic inheritance (MAT-HET, born from WT-dams × KO-fathers; MIX-HET, born from HET-dams × KO-fathers). As controls, we used both wild-type (WT) rats and their socially-isolated (WTi) siblings. We ran the schedule-induced polydipsia (SIP) protocol, to induce compulsive behavior; then the Y-maze and marble-burying tests, to verify its actual development. Only MAT-HET (who inherited the functional DAT allele from the WT mother) is vulnerable to developing compulsive behavior. MAT-HET rats drank increasingly more water during SIP; they showed significant perseverance in the Y-maze test and exhibited compulsive actions in the marble-burying test. Interestingly, compulsive behaviors of MAT-HET rats correlated with expression ex vivo of different genes in different areas. Regarding the prefrontal cortex (PFC), D2R correlated with Y-maze "perseverance" in addition to BDNF; considering the amygdala (AMY), both D3R and OXTR correlated with SIP "licks." Indeed, compulsivity may be linked to D2R and BDNF in PFC, while extreme anxiety in MAT-HET rats may be associated with D3R and OXTR in the AMY. These results confirm some similarities between MAT-HET and DAT-KO subjects, and link the epigenetic context of the DAT gene to the development of compulsive behavior.

Behavioral Phenotype in Heterozygous DAT Rats: Transgenerational Transmission of Maternal Impact and the Role of Genetic Asset.

Dopamine transporter (DAT) is involved in dopamine (DA) reuptake in presynaptic terminals. Deletion of DAT results in a hyperdopaminergic KO-rat phenotype. To conduct our studies in heterozygous DAT rats, several pedigree lines were created, with known derivation of the allele (i.e., maternal or paternal). Our purpose was to elucidate the role of parental origin rather than maternal care, assessing if maternal maltreatments generated sequelae in female offspring. In the first experiment, female rats and their pups were observed during postnatal lactation. Control dams were WT and heterozygous ones were MAT (but K-MAT, with previous experience of early maltreatment by their KO adoptive dams). WT dams were highly attracted to their offspring (predictably, they spent a lot of time licking their pups); in contrast, K-MAT dams showed strangely comparable levels of caring for their pups and exploring the environment. Subsequently, peculiar features of the circadian cycle were found in adolescent rats with different epigenotypes (WT, MUX = offspring of MAT father, MIK = offspring of K-MAT dam). The MIK epigenotype produced locomotor hyperactivity also during resting hours, well above typical values. The MUX epigenotype, on the other hand, was less active and presented a depression-like profile. This study is unique: maltreatment was generated in a spontaneous way from a DAT-KO mother to offspring. We highlight how future studies will address separate contributions by genotype and upbringing. In conclusion, paternal-allele asset generates sequelae diametrically opposed to the inheritance of early maternal trauma.

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.

Dopaminergic modulation of the circadian activity and sociability: Dissecting parental inheritance versus maternal styles as determinants of epigenetic influence.

In mammalians, social life and circadian rhythms find their neurobiological basis in a network that includes the dopaminergic system. The malfunctioning of dopamine pathways can lead to various disorders such as Attention-Deficit/Hyperactivity and Obsessive/compulsive disorders. A useful research approach is to exploit animal models that carry a functional silencing of SLC6A3 gene, encoding the dopamine transporter (DAT). Hyperactivity, working memory deficits, and asocial tendencies are core features in truncated-DAT rats, for example. We investigated how inheritance and maternal caring style influence circadian rhythms and social behaviours in DAT heterozygous (HET) rats, belonging to four groups: Mat-P, Mat-M, Mix-P, and Mix-M (Mat label stands for care from wild-type dam, Mix label stands for care by heterozygous dam; M label stands for maternal wild-DAT and P label stands for paternal wild-DAT). In Experiment 1, we monitored 24/7 the spontaneous locomotor activity of peri-adolescent subjects. Hyperactivity occurred only in P-asset subjects (with maternal-origin truncated-DAT allele) at specific bins of the day. In Experiment 2, we observed social interactions of the same rats. Mix-M subjects (raised by HET dams and/or inheriting the wild-DAT allele from mothers) tend to interact with all rats; Mat-P (cared by WT dams and/or inheriting the truncated-DAT allele from mothers) seem to be ignored, when acting as stimulus subjects. Overall, results confirm complex modulations for circadian cycle and social life: flexible DAT expression in HET subjects depends on epigenetic combinations of parental inheritance and early experiential factors. Once confirmed, these data could shed light on trans-generational contributions to dopaminergic-related disorders.

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.

Altering the development of the dopaminergic system through social play in rats: Implications for anxiety, depression, hyperactivity, and compulsivity.

Dopamine is essential to many functions like reward, motivation, and attention; when its neural pathways do not function properly, various disorders (e.g., anxiety, depression, hyperactivity, compulsions) can arise. Truncated-DAT rats display persistent stereotypies and aggressiveness; hence they are a new valuable animal model to study the pathogenesis of these disorders. The focus of research is often on the individual epigenetic determinants and much less on the impact of social experiences. Here, we investigate the developmental impact of the social environment on adolescent wild type (WT) rats. We divided subjects at weaning into three groups: living with another adolescent (WT Peer), with a WT adult, or with a truncated-DAT one, and we observed homecage social behavior of these pairs (play, jump, victory, and "bullying") during whole adolescence. When adult, we observed the same subjects in plus maze, forced swim, and social preference tests to measure levels of anxiety, depression, and quality of social interactions. Compared to the other groups, WT rats that had spent their adolescence with a truncated-DAT adult as companion show more anxious, depressive, hyperactive, impulsive, and compulsive behaviours. Results confirm that social interactions and healthy play (i.e., when play has behavioural, social, and psychomotor rewards that support the cognitive, emotional and physical development of the individual) are essential to neurobehavioral maturation. Conversely, anomalous interactions like poor play and "bullying" in developing rats may impact onto their dopaminergic system. Consequently, an impoverished social play could be one of the factors contributing to the appearance of putative indexes of attention deficit hyperactivity disorder (ADHD) and\or obsessive-compulsive disorder (OCD).

"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.

Epigenetic regulation of DAT gene promoter modulates the risk of externalizing and internalizing behaviors on a normative population: An explorative study.

Accumulating research addressed epigenetic modifications and their role on behavioral phenotypes. We recently proposed to study methylation dynamics of two CpG motifs within the 5'-UTR of dopamine transporter (DAT) gene. Starting from a normative population sample of young adults, we selected three sub-groups based on their prevalent symptoms: subjects were assigned to Internalizing, Externalizing and Low-risk sub-groups according to elevated scores in specific phenotypic scales. Using a new approach, we calculated three independent matrixes of cross-correlation between CpG methylation levels, one within each phenotypic sub-group, to determine in which dynamics did the sub-groups differ. We found specific cross-correlation patterns in Externalizing (CpG1, 2 and 3, opposite to the methylation at CpG6) and Internalizing individuals (CpG1 methylation opposite to CpG2, 3 and 6), while Low-risk individuals could follow both trends. The aim of our study was to look for a specific DAT methylation pattern, providing a biomarker that allows early identification of the risk for psycho-pathological deviance.