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Effects of restraint stress on functional and structural components of the pituitary gland. (A) Exposure to restraint stress significantly reduced levels of GM130 in control rats (DAT +/+);
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The activity of the hypothalamus–pituitary–adrenal (HPA) axis is pivotal in homeostasis and presides the adaptative response to stress. Dopamine Transporter (DAT) plays a key role in the regulation of the HPA axis. We used young adult female DAT Knockout (KO) rats to assess the effects of DAT ablation (partial, heterozygous DAT+/-, or total, homozy...
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... Blot data presented in Figure 5, Figure 6, Figure 7, Figure 8, and supp. Figure 1 show the comparison between basal condition and stress. ...
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... DAT+/+ control rats showed a significant decrease (~2-fold), GM130 was not significantly altered after stress in DAT+/-rats. On the contrary, DAT-/-rats showed a significant increase (~2-fold) in GM130 levels after restraint stress (Unpaired t-test, two-tailed, p = 0.0001 DAT+/+ control vs stress; p = 0.0977 DAT+/-control vs stress; p = 0.0161 DAT-/-control vs stress, ( Figure 6A and 6B). Moreover, the levels of the posterior lobe marker Cyclin D1 was significantly reduced after restraint stress in both DAT+/+ and DAT+/-rats, while its levels significantly increased (~2-fold) in the pituitary gland of DAT-/-rats (Unpaired t-test, two-tailed, p = 0.0352 DAT+/+ control vs stress; p = 0.0114 DAT+/-control vs stress; p = 0.0035 DAT-/-control vs stress, Figure 6C and 6D). ...
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... the contrary, DAT-/-rats showed a significant increase (~2-fold) in GM130 levels after restraint stress (Unpaired t-test, two-tailed, p = 0.0001 DAT+/+ control vs stress; p = 0.0977 DAT+/-control vs stress; p = 0.0161 DAT-/-control vs stress, ( Figure 6A and 6B). Moreover, the levels of the posterior lobe marker Cyclin D1 was significantly reduced after restraint stress in both DAT+/+ and DAT+/-rats, while its levels significantly increased (~2-fold) in the pituitary gland of DAT-/-rats (Unpaired t-test, two-tailed, p = 0.0352 DAT+/+ control vs stress; p = 0.0114 DAT+/-control vs stress; p = 0.0035 DAT-/-control vs stress, Figure 6C and 6D). ...
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... then analyzed whether TCF4 and NCAM were affected by stress. In both cases, the levels of these proteins were significantly higher in DAT-/-rats after restraint, with a ~2-fold increase for TCF4 (Unpaired t-test, two-tailed, p = 0.6662 DAT+/+ control vs stress; p = 0.5829 DAT+/-control vs stress; p = 0.0087 DAT-/-control vs stress) and ~3-fold increase for NCAM, respectively (Unpaired t-test, two-tailed, p = 0.0986 DAT+/+ control vs stress; p = 0.4514 DAT+/-control vs stress; p = 0.0001 DAT-/-control vs stress, Figure 6E, 6F, 6G, and 6H). Restraint stress did not affect the levels of these proteins in both DAT+/+ and DAT +/-rats. ...
Citations
... 43,44 Prior work in DAT+/À rats demonstrate in home cage, or in long or repeated sessions in the open field, that general locomotor activity does not differ from DAT+/+ animals. 23,45 Here, we focus on initial locomotor activity in a novel arena, and we provide a direct link between response to novelty and ethanol consumption in rodents. Interestingly, the relationship between the locomotor response to a novel environment and binge-like drinking, though significant, appears to be weaker in DAT +/À males than in wild-type counterparts. ...
Mesolimbic dopamine signaling plays a major role in alcohol and substance use disorders as well as comorbidities such as anxiety and depression. Growing evidence suggests that alcohol drinking is modulated by the function of the dopamine transporter (DAT), which tightly regulates extracellular dopamine concentrations. Adult male rats on a Wistar Han background (DAT+/+) and rats with a partial DAT deletion (DAT+/-) were used in this study. First, using fast-scan cyclic voltammetry in brain slices containing the nucleus accumbens core from ethanol-naïve subjects, we measured greater evoked dopamine concentrations and slower dopamine reuptake in DAT+/- rats, consistent with increased dopamine signaling. Next, we measured ethanol drinking using the intermittent access two-bottle choice paradigm (20% v/v ethanol vs. water) across 5 weeks. DAT+/- rats voluntarily consumed less ethanol during its initial availability (the first 30 min), especially after longer periods of deprivation. In addition, DAT+/- males consumed less ethanol that was adulterated with the bitter tastant quinine. These findings suggest that partial DAT blockade and concomitant increase in brain dopamine levels has potential to reduce drinking and ameliorate alcohol use disorder (AUD).
... Notably, the selective decrease of DAT expression in NAcc of adult mice resulted in the same behavioral changes [85]. It should also be considered that ablation of DAT is associated with altered behavioral reactions to stress [86]. It should also be noticed that some scientific groups reported controversial results regarding this topic. ...
The key element of dopamine (DA) neurotransmission is undoubtedly DA transporter (DAT), a transmembrane protein responsible for the synaptic reuptake of the mediator. Changes in DAT’s function can be a key mechanism of pathological conditions associated with hyperdopaminergia. The first strain of gene-modified rodents with a lack of DAT were created more than 25 years ago. Such animals are characterized by increased levels of striatal DA, resulting in locomotor hyperactivity, increased levels of motor stereotypes, cognitive deficits, and other behavioral abnormalities. The administration of dopaminergic and pharmacological agents affecting other neurotransmitter systems can mitigate those abnormalities. The main purpose of this review is to systematize and analyze (1) known data on the consequences of changes in DAT expression in experimental animals, (2) results of pharmacological studies in these animals, and (3) to estimate the validity of animals lacking DAT as models for discovering new treatments of DA-related disorders.
... KO male and female rats showed acute temporary reduced locomotor activity after ip administration, which could have resulted from the stress generated by the hand restraint needed for the administration. The vulnerability to the restraint and handling for ip administration agrees with recent data showing increased response to stress in these rats [50,51] and relevant alterations in brain areas relevant for impulsivity and behavioral control (prefrontal cortex) during vulnerable developmental stages such as adolescence [52]. ...
... However, additional information is needed regarding DAT's role and the impact that genetic DAT perturbation can have on several dopaminergic functions based on sex differences [55]. Importantly, the rate of comorbidity between drug abuse and anxiety disorders is higher in women than men [83], which gives support to the relation between our current results and our previous study demonstrating that HET female rats are more vulnerable to stress effects [51]. ...
... Rats were genotyped at postnatal day (PND) 18-21 as previously described [19]. We recently confirmed reduced striatal protein DAT levels in HET as well as lack of DAT in KO rats [51], Animals were weaned at PND 21-23. Animals were housed in groups after weaning in a temperature (21 ± 1 • C) and humidity-controlled environment under a 12 h light/dark cycle. ...
Cocaine is a powerful psychostimulant that is one of the most widely used illicit addictive. The dopamine transporter (DAT) plays a major role in mediating cocaine’s reward effect. Decreases in DAT expression increase rates of drug abuse and vulnerability to comorbid psychiatric disorders. We used the novel DAT transgenic rat model to study the effects of cocaine on locomotor behaviors in adolescent rats, with an emphasis on sex. Female rats showed higher response rates to cocaine at lower acute and chronic doses, highlighting a higher vulnerability and perceived gender effects. In contrast, locomotor responses to an acute high dose of cocaine were more marked and sustained in male DAT heterozygous (HET) adolescents. The results demonstrate the augmented effects of chronic cocaine in HET DAT adolescent female rats. Knockout (KO) DAT led to a level of hyperdopaminergia which caused a marked basal hyperactivity that was unchanged, consistent with a possible ceiling effect. We suggest a role of alpha synuclein (α-syn) and PICK 1 protein expressions to the increased vulnerability in female rats. These proteins showed a lower expression in female HET and KO rats. This study highlights gender differences associated with mutations which affect DAT expression and can increase susceptibility to cocaine abuse in adolescence.
... Dopamine transporter has been shown to par ticipate in the regulation of the activity of the hypothalamus-pituitary-adrenal cortex axis at both the central and peripheral levels. rats show minor changes in pituitary homeostatic mechanisms that lead to increased vulnerability to stress in female rats with partial deletion of DAT [25]. Learning in the Morris Water maze is accompanied by stress. ...
... В 2018 г. с помощью метода редактирования генома с использованием нуклеаз с «цинковыми пальцами» (zinc fingers) были созданы крысы, нокаутные по гену Slc6a3 -DAT-knockout-(DAT-KO) крысы, которые отличаются высокой внеклеточной концентрацией дофамина в полосатом теле [19,20] и характерным фенотипом. Как и созданные ранее DAT-KO-мыши [21], DAT-KO-крысы имеют меньшую массу тела по сравнению с гетерозиготами (heterozygote type, HT) и особями «дикого типа» (wild type, WT), им свойственна выраженная двигательная гиперактивность и различные стереотипии [19,22]. Установлено также, что мутантные крысы хуже справляются с задачами для оценки рабочей памяти [9] и визуального обучения [23]. ...
Introduction . Changes in the expression of the dopamine transporter (DAT) have been identified in patients with a number of neuropsychiatric disorders, but their significance for pathogenesis remains unclear. DAT knockout rats are a promising model of frontostriatal dysfunctions involved in adapting learning processes to current organism's needs, motivation, and experience.
The objective was to evaluate the effect of DAT disruption on the interaction of classical and instrumental conditioning processes (associative learning).
Methods and materials . Rats from a local colony were used: knockouts (n=31), heterozygotes (n=32), and wild type rats (n=24). DAT knockout ( Slc6a3 ) was detected by PCR followed by restriction analysis. The rats were kept individually with limited access to food and unlimited access to water. In the operant chambers equipped with a MED-PC interface (MED Associates, USA), 3 experiments were performed: 1) classical conditioning; 2) autoshaping of operant responding; 3) reward learning based on secondary reinforcement. Statistical analysis was performed using SigmaPlot 12.5 (Systat Software Inc., USA) and SPSS Statistics 21 (IBM, USA).
Results . We revealed that DAT disruption in rats did not affect the formation of conditioned reflex connections in classical conditioning (1) but was accompanied by impairments in the autoshaping of the operant response (2) and learning based on secondary reinforcement (3).
Conclusion . The observed impairments of associative learning might be associated with a decreased incentive value of stimuli in hyperdopaminergic state.
... Traditionally, center avoidance was considered in the context of anxiety, however behavioral data in DAT-KO animals in this regard are unclear. While higher corticosterone levels and increased stress susceptibility in female DAT-KO rats [50] support this notion, reduced freezing in males exposed to fear-associated context [18] questions this idea. It appears more probable that center avoidance adds to a series of compulsive-like features in DAT- ) robustness against random attacks; Small-World Prop. ...
... The results of our study are limited to male rats. However, based on recent evidence of comparable behavioral phenotypes in female DAT-KO rats [50,72] and of higher DAT activity and striatal dopamine release in female rats in general [73], we would expect analogous or conceivably even more severe effects induced by greater dopaminergic excitotoxicity. ...
Silencing of dopamine transporter (DAT), a main controlling factor of dopaminergic signaling, results in biochemical and behavioral features characteristic for neuropsychiatric diseases with presumed hyperdopaminergia including schizophrenia, attention deficit hyperactivity disorder (ADHD), bipolar disorder, and obsessive-compulsive disorder (OCD). Investigation of DAT silencing thus provides a transdiagnostic approach towards a systems-level understanding of common underlying pathways. Using a high-field multimodal imaging approach and a highly sensitive cryogenic coil, we integrated structural, functional and metabolic investigations in tandem with behavioral assessments on a newly developed preclinical rat model, comparing DAT homozygous knockout (DAT-KO, N = 14), heterozygous knockout (N = 8) and wild-type male rats (N = 14). We identified spatially distributed structural and functional brain alterations encompassing motor, limbic and associative loops that demonstrated strong behavioral relevance and were highly consistent across imaging modalities. DAT-KO rats manifested pronounced volume loss in the dorsal striatum, negatively correlating with cerebellar volume increase. These alterations were associated with hyperlocomotion, repetitive behavior and loss of efficient functional small-world organization. Further, prefrontal and midbrain regions manifested opposite changes in functional connectivity and local network topology. These prefrontal disturbances were corroborated by elevated myo-inositol levels and increased volume. To conclude, our imaging genetics approach provides multimodal evidence for prefrontal-midbrain decoupling and striato-cerebellar neuroplastic compensation as two key features of constitutive DAT blockade, proposing them as transdiagnostic mechanisms of hyperdopaminergia. Thus, our study connects developmental DAT blockade to systems-level brain changes, underlying impaired action inhibition control and resulting in motor hyperactivity and compulsive-like features relevant for ADHD, schizophrenia and OCD.
... Peculiar phenotypic features are linked to the changes in DA levels. Comparable to DAT-KO mice (21), DAT-KO rats weigh less than heterozygote (HT) and wild-type (WT) rats, demonstrating dramatically increased locomotor activity and prominent motor and oral stereotypies (19,22). Mutant rats also display impaired working memory (8), changes in learning an object recognition task (23), and altered male sexual behavior (24). ...
Dopamine (DA) is critically involved in different functions of the central nervous system (CNS) including control of voluntary movement, affect, reward, sleep, and cognition. One of the key components of DA neurotransmission is DA reuptake by the DA transporter (DAT), ensuring rapid clearance of DA from the synaptic cleft. Thus, lack of DAT leads to persistent high extracellular DA levels. While there is strong evidence for a role of striatal dopaminergic activity in learning and memory processes, little is known about the contribution of DAT deficiency to conditional learning impairments and underlying molecular processes. DAT-knockout (DAT-KO) rats were tested in a set of behavioral experiments evaluating conditional associative learning, which requires unaltered striatal function. In parallel, a large-scale proteomic analysis of the striatum was performed to identify molecular factors probably underlying behavioral patterns. DAT-KO rats were incapable to acquire a new operant skill in Pavlovian/instrumental autoshaping, although the conditional stimulus–unconditional stimulus (CS-US) association seems to be unaffected. These findings suggest that DAT directly or indirectly contributes to the reduction of transference of incentive salience from the reward to the CS. We propose that specific impairment of conditional learning might be caused by molecular adaptations to the hyperdopaminergic state, presumably by dopamine receptor 1 (DRD1) hypofunction, as proposed by proteomic analysis. Whether DRD1 downregulation can cause cognitive deficits in the hyperdopaminergic state is the subject of discussion, and further studies are needed to answer this question. This study may be useful for the interpretation of previous and the design of future studies in the dopamine field.
... The G protein-independent D2R signaling is represented by βArr2-mediated signaling. The activation of the D2-like receptors contributes to the constitution of a protein complex composed of protein phosphatase 2A (PP2A), serine/threonine kinase (Akt), and βArr2, where PP2A increases the dephosphorylation and inactivation of Akt, leading to the modulation of glycogen synthase kinase-3 (GSK-3) activation Particularly, recent studies of Illiano et al. (2020) showed that in rodents, the lack of DAT results in increased vulnerability and aberrant autonomic response to acute stress. In particular, DAT represents a preferential target for parkinsonian neurotoxins, as the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), MPP + , is specifically transported by DAT and concentrated within the nigral DA neurons where it inhibits complex I of the mitochondrial electron transport chain (METC), resulting in ATP depletion and subsequent neuronal cell death (Di Monte & Langston, 1995;Langston, 2017;Schildknecht et al., 2017) (Figure 3). ...
Dopamine (DA) signaling via G protein-coupled receptors is a multifunctional neurotransmitter and neuroendocrine-immune modulator. The DA nigrostriatal pathway, which controls the motor coordination, progressively degenerates in Parkinson's disease (PD), a most common neurodegenerative disorder (ND) characterized by a selective, age-dependent loss of substantia nigra pars compacta (SNpc) neurons, where DA itself is a primary source of oxidative stress and mitochondrial impairment, intersecting astrocyte and microglial inflammatory networks. Importantly, glia acts as a preferential neuroendocrine-immune DA target, in turn, counter-modulating inflammatory processes. With a major focus on DA intersection within the astrocyte-microglial inflammatory network in PD vulnerability, we herein first summarize the characteristics of DA signaling systems, the propensity of DA neurons to oxidative stress, and glial inflammatory triggers dictating the vulnerability to PD. Reciprocally, DA modulation of astrocytes and microglial reactivity, coupled to the synergic impact of gene-environment interactions, then constitute a further level of control regulating midbrain DA neuron (mDAn) survival/death. Not surprisingly, within this circuitry, DA converges to modulate nuclear factor erythroid 2-like 2 (Nrf2), the master regulator of cellular defense against oxidative stress and inflammation, and Wingless (Wnt)/β-catenin signaling, a key pathway for mDAn neurogenesis, neuroprotection, and immunomodulation, adding to the already complex "signaling puzzle," a novel actor in mDAn-glial regulatory machinery. Here, we propose an autoregulatory feedback system allowing DA to act as an endogenous Nrf2/Wnt innate modulator and trace the importance of DA receptor agonists applied to the clinic as immune modifiers.
... However, little research has focused on sex-specific studies of these pathologies at both clinical [4,6] and preclinical level [7,8]. Recent studies focusing on adult dopamine transporter deletion in rats, in the heterozygous and homozygote genotypes, highlighted that female adult rats from both genotypes display model validity for PTSD and ADHD [9]. On the other hand, male adult DAT−/− rats also display model validity for ADHD [10,11]. ...
... on-6 p.m. off, 21 ± 1 • C, 40-70% humidity). The breeding scheme was performed as reported from our group, mating mature DAT+/− female rats of fertile age (>2 months old) with mature DAT+/− male rats [9,30]. We acknowledge the possible effect that the breeding strategy could have on the study [30], out of our scope of work. ...
... Western blotting experiments were performed, as previously described [9], with minor modifications. Prelimbic PFC tissue was dissected from one hemisphere and mechanically homogenized in extraction buffer (20 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 1% Triton-X-100, 1 mmol/L EDTA, 1 mmol/L EGTA) supplemented with PhosSTOP™ (Millipore Sigma #4906837001) and Complete Mini, EDTA-free (Roche, 11836170001). ...
Monoamine dysfunctions in the prefrontal cortex (PFC) can contribute to diverse neuropsychiatric disorders, including ADHD, bipolar disorder, PTSD and depression. Disrupted dopamine (DA) homeostasis, and more specifically dopamine transporter (DAT) alterations, have been reported in a variety of psychiatric and neurodegenerative disorders. Recent studies using female adult rats heterozygous (DAT+/−) and homozygous (DAT−/−) for DAT gene, showed the utility of those rats in the study of PTSD and ADHD. Currently, a gap in the knowledge of these disorders affecting adolescent females still represents a major limit for the development of appropriate treatments. The present work focuses on the characterization of the PFC function under conditions of heterozygous and homozygous ablation of DAT during early adolescence based on the known implication of DAT and PFC DA in psychopathology during adolescence. We report herein that genetic ablation of DAT in the early adolescent PFC of female rats leads to changes in neuronal and glial cell homeostasis. In brief, we observed a concurrent hyperactive phenotype, accompanied by PFC alterations in glutamatergic neurotransmission, signs of neurodegeneration and glial activation in DAT-ablated rats. The present study provides further understanding of underlying neuroinflammatory pathological processes that occur in DAT-ablated female rats, what can provide novel investigational approaches in human diseases.
