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.

Keeping Track of the Genealogy of Heterozygotes Using Epigenetic Reference Codes and Breeding Tables.

Studying neurobehavioral consequences of the hypofunctional dopamine transporter (DAT) across several generations entails the need to monitor allelic transmission to offspring, taking into account both maternal and paternal inheritance. Since each type of heterozygote expresses differential phenotypes, based on lineage of inheritance for wild and mutated alleles (from male or female ancestors), it is important to track transgenerational epigenetic effects. We deemed it essential to assign specific abbreviations identifying their characteristics. Therefore, we devised a Mendelian-inspired table to keep track of these. Starting from two progenitors (WT and KO) we named resulting heterozygous progenies MAT and PAT to differentiate them based on inheritance of the wild allele (from the mother or father). Tracing subsequent generations, similar logic has been followed: if coupling HET dams with KO males, initials "M" [(grand)maternal] and "P" [(grand)paternal] are kept, but "AT" is turned into "IX" (MIX and PIX), while if breeding HETs with WTs, "M" is changed to "W" resembling an upside down "M" and "P" to "S" for "sperm" (WAT and SOT). To underline the development within "hyperdopaminergic-uterus" a central letter "U" is added (MUX, PUX, and QULL), while a Greek initial (µAT, µIX, and νIX) underlines the uterine-worsened origin of the allele. In HET × HET breeding (GIX and DIX), the mutated allele can be inherited from both sides of the genealogical line. However, when the mother is MAT, wild and mutated alleles encounter for the first time, causing putative anomalies in the progeny. Replacing dam with a second-generation female (MIX and MUX) may mitigate epigenetic effects on third-generation offspring; therefore suffixes ("-f," "-fu," "-ϕ," and "-ϕu") emphasize that subsequent-generation dams imply that the alleles already encountered in HET (rather than WT) grand-dams.

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.