Own or dam's genotype? Classical colony breeding may bias spontaneous and stress-challenged activity in DAT-mutant rats.

There is considerable interest in understanding what makes an individual vulnerable or resilient to the deleterious effects of stressful events. From candidate genes, dopamine (DA) and dopamine transporter (DAT) have been linked to anxiety, depression, and post-traumatic stress disorder. We investigated role of DAT using the new DAT heterozygous (DAT-HET) and homozygous mutant (DAT-KO) rat models of hyperdopaminergia. We studied the impact of two breeding conditions in spontaneous locomotor behavior of female rats. The classical colony, through mating DAT-HET males × DAT-HET females (breeding HET-HET), was used. A second WT colony was derived and maintained (breeding WT-WT). Additionally, a subgroup of rats was bred through mating DAT-KO males × WT females (atypical HET, breeding KO-WT). We studied the effects of genotype and its interaction with maternal care (depending by breeding condition). HET-HET breeding led to reduced activity in HET females compared to WT rats (from WT-WT breeding). However, HET females from KO-WT breeding did not differ so much from WT rats (WT-WT breeding). The maternal-care impact was then confirmed: HET mothers (breeding HET-HET) showed reduced liking/grooming of pups and increased digging away from nest, compared to WT mothers (breeding WT-WT). In their female offspring (HET, breeding HET-HET vs. WT, breeding WT-WT), isolation plus wet bedding induced higher and more persistent impact on activity of HET rats, even when the stressor was removed. Our results highlight the importance of epigenetic factors (e.g., maternal care) in responses to stress expressed by offspring at adulthood, quite independently of genotype. DAT hypofunction could determinate vulnerability to stressful agents via altered maternal care.

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.

A new "sudden fright paradigm" to explore the role of (epi)genetic modulations of the DAT gene in fear-induced avoidance behavior.

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.

Motor Transitions' Peculiarity of Heterozygous DAT Rats When Offspring of an Unconventional KOxWT Mating.

Causal factors of psychiatric diseases are unclear, due to gene × environment interactions. Evaluation of consequences, after a dopamine-transporter (DAT) gene knock-out (DAT-KO), has enhanced our understanding into the pathological dynamics of several brain disorders, such as Attention-Deficit/Hyperactivity and Bipolar-Affective disorders. Recently, our attention has shifted to DAT hypo-functional (heterozygous, HET) rodents: HET dams display less maternal care and HET females display marked hypo-locomotion if cared by HET dams (Mariano et al., 2019). We assessed phenotypes of male DAT-heterozygous rats as a function of their parents: we compared "maternal" origin (MAT-HET, obtained by breeding KO-male rats with WT-female dams) to "mixed" origin (MIX-HET, obtained by classical breeding, both heterozygous parents) of the allele. MAT-HET subjects had significantly longer rhythms of daily locomotor activity than MIX-HET and WT-control subjects. Furthermore, acute methylphenidate (MPH: 0, 1, 2 mg/kg) revealed elevated threshold for locomotor stimulation in MAT-HETs, with no response to the lower dose. Finally, by Porsolt-Test, MAT-HETs showed enhanced escape-seeking (diving) with more transitions towards behavioral despair (floating). When comparing both MAT- and MIX-HET to WT-control rats, decreased levels of DAT and HDAC4 were evident in the ventral-striatum; moreover, with respect to MIX-HET subjects, MAT-HET ones displayed increased DAT density in dorsal-striatum. MAT-HET rats displayed region-specific changes in DAT expression, compared to "classical" MIX-HET subjects: greater DAT availability may elevate threshold for dopamine action. Further behavioral and epigenetic characterizations of MAT-HETs, together with deeper characterization of maternal roles, could help to explore parent-of-origin mechanisms for such a peculiar phenotype.

Behavioral characterization of DAT-KO rats and evidence of asocial-like phenotypes in DAT-HET rats: The potential involvement of norepinephrine system.

Dopamine (DA) is a key neurotransmitter of the central nervous system, whose availability is regulated by the dopamine transporter (DAT). Deletion of DAT gene leading to hyperdopaminergia was previously performed on mouse models. This enabled recapitulation of the core symptoms of Attention-Deficit / Hyper-activity Disorder (ADHD), which include hyperactivity, inattention and cognitive impairment. We used recently developed DAT knockout (DAT-KO) rats to carry out further behavioral profiling on this novel model of hyperdopaminergia. DAT-KO rats display elevated locomotor activity and restless environmental exploration, associated with a transient anxiety profile. Furthermore, these rats show pronounced stereotypy and compulsive-like behavior at the Marble-Burying test. Homozygous DAT-KO rats mantain intact social interaction when tested in a social-preference task, while heterozygous (HET) rats show high inactivity associated with close proximity to the social stimulus. Ex-vivo evaluation of brain catecholamines highlighted increased levels of norepinephrine in the hippocampus and hypothalamus exclusively of heterozygous rats. Taken together, our data present evidence of unexpected asocial tendencies in heterozygous (DAT-HET) rats associated with neurochemical alterations in norepinephrine neurotransmission. We shed light on the behavioral and neurochemical consequences of altered DAT function in a higher, more complex model of hyperdopaminergia. Unraveling the role of DA neurotransmission in DAT-KO rats has very important implications in the understanding of many psychiatric illnesses, including ADHD, where alterations in DA system have been demonstrated.

Prior Activation of 5-HT7 Receptors Modulates the Conditioned Place Preference With Methylphenidate.

The serotonin receptor subtype 7 (5-HT7R) is clearly involved in behavioral functions such as learning/memory, mood regulation and circadian rhythm. Recent discoveries proposed modulatory physiological roles for serotonergic systems in reward-guided behavior. However, the interplay between serotonin (5-HT) and dopamine (DA) in reward-related behavioral adaptations needs to be further assessed. TP-22 is a recently developed arylpiperazine-based 5-HT7R agonist, which is also showing high affinity and selectivity towards D1 receptors. Here, we report that TP-22 displays D1 receptor antagonist activity. Moreover, we describe the first in vivo tests with TP-22: first, a pilot experiment (assessing dosage and timing of action) identified the 0.25 mg/kg i.v. dosage for locomotor stimulation of rats. Then, a conditioned place preference (CPP) test with the DA-releasing psychostimulant drug, methylphenidate (MPH), involved three rat groups: prior i.v. administration of TP-22 (0.25 mg/kg), or vehicle (VEH), 90 min before MPH (5 mg/kg), was intended for modulation of conditioning to the white chamber (saline associated to the black chamber); control group (SAL) was conditioned with saline in both chambers. Prior TP-22 further increased the stimulant effect of MPH on locomotor activity. During the place-conditioning test, drug-free activity of TP-22+MPH subjects remained steadily elevated, while VEH+MPH subjects showed a decline. Finally, after a priming injection of TP-22 in MPH-free conditions, rats showed a high preference for the MPH-associated white chamber, which conversely had vanished in VEH-primed MPH-conditioned subjects. Overall, the interaction between MPH and pre-treatment with TP-22 seems to improve both locomotor stimulation and the conditioning of motivational drives to environmental cues. Together with recent studies, a main modulatory role of 5-HT7R for the processing of rewards can be suggested. In the present study, TP-22 proved to be a useful psychoactive tool to better elucidate the role of 5-HT7R and its interplay with DA in reward-related behavior.

Activation of 5-HT7 receptor by administration of its selective agonist, LP-211, modifies explorative-curiosity behavior in rats in two paradigms which differ in visuospatial parameters.

AIMS: The serotonin 7 receptor (5-HT7R) subtype, coded by Htr7 gene, is broadly expressed in the central nervous system (CNS) with clear involvement in behavioral functions such as learning/memory, regulation of mood, and circadian rhythms. In this study, we assessed effects of 5-HT7R stimulation by administration of its selective agonist, LP-211 (0.25 mg/kg i.p.), in adult Wistar-Han rats. METHODS: We used two different explorative-curiosity tests. Drug was administered either before one side-chamber familiarization (CF/V group) or immediately after it, to act on consolidation of familiarization (V/CF group). RESULTS: Exp. 1 for novelty seeking in black/white boxes (BWB), with door opening after 5 minutes in the familiar chamber, showed that (i) time spent in the novel environment (significantly higher than in familiar chamber for controls) is enhanced in V/CF group (potentiated recognition for a "visual" consolidation) and not different in CF/V group; (ii) activity and chamber transitions, made by CF/V rats, are significantly higher than for other groups (interference on recognition for a "spatial" acquisition). Exp. 2 for novelty preference in D- vs L-shaped chambers (D/L), with start from neutral center, gave different results: (i) time spent in the novel environment by CF/V group is significantly higher than other groups (potentiated "cognitive" acquisition); (ii) chamber transitions made by V/CF group are significantly higher than other groups (potentiated "emotional" consolidation). CONCLUSION: These apparently conflicting results may reflect LP-211 effects on visual vs spatial memory (D/L apparatus has more pronounced hippocampal components than BWB). However, further experiments are needed to analyze more in depth the mechanisms involved.