Alterations in dopamine (DA) reuptake are involved in several psychiatric disorders whose symptoms can be investigated in knock out rats for the DA transporter (DAT‐KO). Recent studies evidenced the role of epigenetic DAT modulation in depressive‐like behavior. Accordingly, we used heterozygous (HET) rats born from both HET parents (termed MIX‐HET), compared to HET rats born from WT‐mother and KO‐father (MAT‐HET), implementing the role of maternal care on DAT modulation. We developed a “sudden fright” paradigm (based on dark‐light test) to study reaction to fearful inputs in the DAT‐KO, MAT‐HET, MIX‐HET, and WT groups. Rats could freely explore the whole 3‐chambers apparatus; then, they were gently confined in one room where they experienced the fright; finally, they could freely move again. As expected, after the fearful stimulus only MAT‐HET rats showed a different behavior consisting of avoidance towards the fear‐associated chamber, compared to WT rats. Furthermore, ex‐vivo immuno‐fluorescence reveals higher prefrontal DAT levels in MAT‐HET compared to MIX‐HET and WT rats. Immuno‐fluorescence shows also a different histone deacetylase (HDAC) enzymes concentration. Since HDAC concentration could modulate gene expression, within MAT‐HET fore brain, the enhanced expression of DAT could well impair the corticostriatal‐thalamic circuit, thus causing aberrant avoidance behavior (observed only in MAT‐HET rats). DAT expression seems to be linked to a simply different breeding condition, which points to a reduced care by HET dams for epigenetic regulation. This could imply significant prefronto‐cortical influences onto the emotional processes: hence an excessively frightful response, even to mild stressful agents, may draw developmental trajectories toward anxious and depressed‐like behavior.

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.

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.

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