Deletion Of Dopamine Transporter Research Articles

Patterns of ethanol intake in male rats with partial dopamine transporter deficiency.

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).

Who Knew? Dopamine Transporter Activity Is Critical in Innate and Adaptive Immune Responses.

The dopamine transporter (DAT) regulates the dimension and duration of dopamine transmission. DAT expression, its trafficking, protein-protein interactions, and its activity are conventionally studied in the CNS and within the context of neurological diseases such as Parkinson's Diseases and neuropsychiatric diseases such as drug addiction, attention deficit hyperactivity and autism. However, DAT is also expressed at the plasma membrane of peripheral immune cells such as monocytes, macrophages, T-cells, and B-cells. DAT activity via an autocrine/paracrine signaling loop regulates macrophage responses to immune stimulation. In a recent study, we identified an immunosuppressive function for DAT, where blockade of DAT activity enhanced LPS-mediated production of IL-6, TNF-α, and mitochondrial superoxide levels, demonstrating that DAT activity regulates macrophage immune responses. In the current study, we tested the hypothesis that in the DAT knockout mice, innate and adaptive immunity are perturbed. We found that genetic deletion of DAT (DAT-/-) results in an exaggerated baseline inflammatory phenotype in peripheral circulating myeloid cells. In peritoneal macrophages obtained from DAT-/- mice, we identified increased MHC-II expression and exaggerated phagocytic response to LPS-induced immune stimulation, suppressed T-cell populations at baseline and following systemic endotoxemia and exaggerated memory B cell expansion. In DAT-/- mice, norepinephrine and dopamine levels are increased in spleen and thymus, but not in circulating serum. These findings in conjunction with spleen hypoplasia, increased splenic myeloid cells, and elevated MHC-II expression, in DAT-/- mice further support a critical role for DAT activity in peripheral immunity. While the current study is only focused on identifying the role of DAT in peripheral immunity, our data point to a much broader implication of DAT activity than previously thought. This study is dedicated to the memory of Dr. Marc Caron who has left an indelible mark in the dopamine transporter field.

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.

Rats Lacking Dopamine Transporter Display Increased Vulnerability and Aberrant Autonomic Response to Acute Stress.

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, homozygous DAT-/-) on vulnerability to stress. DAT-/- rats show profound dysregulation of pituitary homeostasis, in the presence of elevated peripheral corticosterone, before and after acute restraint stress. During stress, DAT-/- rats show abnormal autonomic response at either respiratory and cardiovascular level, and delayed body temperature increase. DAT+/- rats display minor changes of hypophyseal homeostatic mechanisms. These rats display a similar pituitary activation to that of the control animals, albeit in the presence of higher release of peripheral corticosterone than DAT-/- after stress, and reduced temperature during stress. Our data indicate that DAT regulates the HPA axis at both the central and peripheral level, including autonomic function during stress. In particular, the partial deletion of DAT results in increased vulnerability to stress in female rats, which display central and peripheral alterations that are reminiscent of PTSD, and they might provide new insights in the pathophysiology of this disorder.

Methamphetamine-induced locomotor activity and sensitization in dopamine transporter and vesicular monoamine transporter 2 double mutant mice

The dopamine transporter (DAT) and the vesicular monoamine transporter 2 (VMAT2) play pivotal roles in the action of methamphetamine (MAP), including acute locomotor effects and behavioral sensitization. However, the relative impact of heterozygous DAT and VMAT2 knockouts (KOs) on the behavioral effects of MAP remains unknown. To evaluate the roles of DAT and VMAT2 in MAP-induced locomotor behavior, we examined locomotor activity and sensitization in heterozygous DAT KO (DAT+/-), heterozygous VMAT2 KO (VMAT2+/-), double heterozygous DAT/VMAT2 KO (DAT+/-VMAT2+/-), and wild-type (WT) mice. Acute 1 mg/kg MAP injection induced significant locomotor increases in WT and VMAT2+/- mice but not in DAT+/- and DAT+/-VMAT2+/- mice. Acute 2 mg/kg MAP significantly increased locomotor activity in all genotypes. Repeated 1 mg/kg MAP injections revealed a delayed and attenuated development of sensitization in DAT+/- and DAT+/-VMAT2+/- mice compared to WT mice and delayed development in VMAT2+/- mice. In repeated 2 mg/kg MAP injections, DAT+/- and DAT+/-VMAT2+/- mice showed delayed but not attenuated development of sensitization, while there was no difference in the onset of sensitization between VMAT2+/- and WT mice. In DAT+/-VMAT2+/- mice, all of MAP-induced behavioral responses were similar to those in DAT+/- but not VMAT2+/- mice. Heterozygous deletion of DAT attenuates the locomotor effects of MAP and may play larger role in behavioral responses to MAP compared to heterozygous deletion of VMAT2.

Transgenic Mouse Studies Reveal Substantial Roles for Opioid Receptors in the Rewarding Effects of Several Classes of Addictive Drugs

In recent years transgenic studies in mice, in particular those utilizing gene knockout techniques, have greatly contributed to our understanding of the molecular mechanisms of drug reward and drug abuse. Many of the initial gene knockouts that have been created focused on the initial molecular targets of drugs of abuse such as the μ opiate receptor (MOR) for morphine, the dopamine transporter (DAT) for cocaine and the vesicular monoamine transporter 2 (VMAT2) for amphetamine. These met with varying degrees of success; for instance, although gene deletion of MOR eliminated virtually all of the effects of morphine, the effects of cocaine were eliminated only by combined deletion of DAT and the serotonin transporter (SERT). However, such studies only address the role of these genes in the actions of a single drug, and not potential contributions to the rewarding and addictive properties of drugs of abuse more generally. Studies of MOR KO mice have been done for a number of addictive drugs and it has become apparent that MOR KO affects the rewarding consequences of a wide range of addictive substances. This contrasts quite sharply with the consequences of other gene deletions, including deletion of the other opiate receptor genes. Furthermore, this conclusion suggests that variants in the MOR gene itself or variation in MOR gene expression may influence susceptibility to drug abuse and provide a common molecular target for treating a wide range of addictions.

Molecular Biology, Pharmacology and Functional Role of the Plasma Membrane Dopamine Transporter

The plasma membrane dopamine transporter (DAT) tightly regulates the extracellular concentrations of dopamine (DA) by re-capturing released neurotransmitter back into the presynaptic neuronal terminals and/or neighboring DA projections thereby providing an effective way to regulate synaptic and extrasynaptic DA levels. This transporter is a primary target of many potent psychotropic drugs and neurotoxins, such as cocaine, amphetamines and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this review we summarize recent advances in understanding the structure, regulation, and functional roles of DAT in normal DA physiology and pathological conditions, such as attention deficit hyperactivity disorder (ADHD) and neurodegenerative processes, as well as their contribution to the pharmacology of psychostimulant drugs. Significant new insights on these issues have been gained using mice with genetic deletion of DAT.

Changes in innate and acquired immune responses in mice with targeted deletion of the dopamine transporter gene

The dopamine transporter (DAT) is responsible for the re-uptake of dopamine into presynaptic nerve terminals and thereby controls dopaminergic neurotransmission. Deletion of DAT results in a hyperdopaminergic phenotype and DAT −/− mice are characterized by pituitary hypoplasia, impaired maternal behavior, and increased locomotion. From earlier studies, we have evidence that the activity of the central dopaminergic system may play a role in determining immune reactivity and disease susceptibility. To further explore the functional relation between the dopaminergic system and the immune system, we investigated the activity of the immune system in DAT −/− mice. We show that in vitro, splenocytes from DAT −/− mice displayed reduced natural killer cell activity and reduced mitogen-induced cytokine responses. In contrast, LPS-induced cytokine production by macrophages was enhanced. In vivo, the cellular response to immunization with ovalbumine (OVA-induced delayed type hypersensitivity response) was significantly reduced. Interestingly, the OVA-induced humoral response (anti-OVA IgG) was increased in DAT −/− mice compared to wild-type animals. Plasma levels of catecholamines and corticosterone did not differ significantly between DAT −/− and wild-type animals. In conclusion, we show in the present study that interfering with the dopaminergic system has major consequences for both the acquired and the innate immune response.

Regional differences in extracellular dopamine and serotonin assessed by in vivo microdialysis in mice lacking dopamine and/or serotonin transporters.

Cocaine conditioned place preference (CPP) is intact in dopamine transporter (DAT) knockout (KO) mice and enhanced in serotonin transporter (SERT) KO mice. However, cocaine CPP is eliminated in double-KO mice with no DAT and either no or one SERT gene copy. To help determine mechanisms underlying these effects, we now report examination of baselines and drug-induced changes of extracellular dopamine (DAex) and serotonin (5-HT(ex)) levels in microdialysates from nucleus accumbens (NAc), caudate putamen (CPu), and prefrontal cortex (PFc) of wild-type, homozygous DAT- or SERT-KO and heterozygous or homozygous DAT/SERT double-KO mice, which are differentially rewarded by cocaine. Cocaine fails to increase DAex in NAc of DAT-KO mice. By contrast, systemic cocaine enhances DAex in both CPu and PFc of DAT-KO mice though local cocaine fails to affect DAex in CPu. Adding SERT to DAT deletion attenuates the cocaine-induced DAex increases found in CPu, but not those found in PFc. The selective SERT blocker fluoxetine increases DAex in CPu of DAT-KO mice, while cocaine and the selective DAT blocker GBR12909 increase 5-HT(ex) in CPu of SERT-KO mice. These data provide evidence that (a) cocaine increases DAex in PFc independently of DAT and that (b), in the absence of SERT, CPu levels of 5-HT(ex) can be increased by blocking DAT. Cocaine-induced alterations in CPu DA levels in DAT-, SERT-, and DAT/SERT double-KO mice appear to provide better correlations with cocaine CPP than cocaine-induced DA level alterations in NAc or PFc.

Dissociation of rewarding and dopamine transporter-mediated properties of amphetamine.

The interaction of amphetamine (AMPH) with the dopamine (DA) transporter (DAT) is thought to be critically important for the DA-elevating actions of this drug. It is commonly believed that DA elevations are involved in the rewarding/reinforcing properties of AMPH and other drugs of abuse. Here, we found that DAT deletion did not eliminate the rewarding effects of AMPH as measured by conditioned place preference (CPP). In fact, mice in which the DAT gene has been deleted (DAT-KO mice) exhibited AMPH-induced CPP for many weeks after the time when extinction occurred in WT mice. Moreover, systemic AMPH still increased extracellular DA in the nucleus accumbens (NAc) of mice lacking the DAT, although local infusion of AMPH into the NAc did not have this effect. By using voltammetry in NAc slices, we found that AMPH did not decrease the rate of DA clearance. The rate of ventral tegmental area DA neuron firing was dramatically inhibited by AMPH in brain slices from WT mice, but there was no inhibition of firing in DAT-KO mice. AMPH-induced CPP was abolished by pretreatment with WAY-100635, a serotonin 5-HT(1A) receptor antagonist, in DAT-KO mice, but the drug did not change AMPH place preference in WT mice. Therefore, despite the absence of the DAT, AMPH displays rewarding effects and causes an increase in extracellular DA in the NAc of DAT-KO mice, acting indirectly in this case. The 5-HT system may be involved in the rewarding effects of AMPH in these mice.

Phosphatidylinositol 3-Kinase, Protein Kinase C, and MEK1/2 Kinase Regulation of Dopamine Transporters (DAT) Require N-terminal DAT Phosphoacceptor Sites

The dopamine transporter (DAT) modulates dopamine neurotransmission and is a primary target for psychostimulant influences on locomotion and reward. Selective DAT expression by dopaminergic neurons has led to use of cocaine analog DAT radioligands to assess rates of progression of dopamine neuronal degeneration in Parkinson's disease. We have documented that DAT is a phosphoprotein that is regulated by phosphorylation through pathways that include protein kinase C cascades. We now extend this work using drugs selective for phosphatidylinositol 3-kinase (PI3K), protein kinase C, MEK1/2, p38 kinase, and Ca2+/calmodulin kinase II. We compare the drug effects on wild type DAT to the effects on 20 DAT mutants and a DAT deletion. PI3K and MEK1/2 modulators exert strong effects on DAT expression patterns and dopamine uptake Vmax. PKC principally modulates Vmax. Neither p38 nor Ca2+/calmodulin kinase II agents exert significant influences on wild type DAT. Several mutants and a DAT with an N-terminal deletion display alterations that interact with the effects of kinase modulators, especially S7A for PKC effects; T62A, S581A, and T612A for PI3K effects; and S12A and T595A mutants for MEK1/2 effects. 32P-Labeling studies confirm several of these effects of kinase pathway modulators on DAT phosphorylation. DAT expression and activities can be regulated by kinase cascades that require phosphoacceptor sites most concentrated in its N terminus. These results have a number of implications for DAT regulation and mandate caution in using DAT radioligand binding to infer changes in dopaminergic neuronal integrity after treatments that alter activities of these kinase pathways.

Decreased Ethanol Preference and Consumption in Dopamine Transporter Female Knock-Out Mice

Background: It is commonly believed that the mesolimbic dopamine (DA) system participates in the etiology of alcoholism. One of the most important regulators of DA synaptic transmission is the DA transporter (DAT). We examined the effects of the genetic reduction or deletion of DAT on voluntary ethanol consumption and ethanol-induced sedation. Methods: Ethanol preference and consumption were assessed in the two-bottle choice paradigm, and the depressant effects of ethanol were evaluated by measuring sleep time after acute injection of ethanol. Results: The latent period to lose the righting reflex was shorter in both knock-out mice (DAT-KO) and heterozygote mice (HET) than in wild-type (WT) mice. No significant difference was found among the three genotypes in the ethanol blood concentration at the onset of regaining the righting reflex. Females of all genotypes consumed more fluid than males of the same genotype. HET and DAT-KO females had increased total fluid consumption compared with WT females. DAT-KO males had increased fluid consumption compared with WT and HET males. Ethanol preference and consumption were not different among male mice of different genotypes. WT and HET females demonstrated significantly higher ethanol consumption than males. HET female mice did not differ from WT mice in ethanol preference. There was no difference between HET and WT mice in the preference for saccharin or quinine solutions. DAT-KO females avoided ethanol, and their consumption and preference were lower than in WT and HET females, despite markedly increased total intake. DAT-KO mice also demonstrated altered taste preference for saccharin and quinine. Conclusions: Partial deletion of DAT results in increased fluid consumption in female mice but does not change ethanol preference in either sex. Complete deletion of DAT reduces ethanol preference in female mice; this may be due to a combination of the pharmacological actions of DAT deletion and alterations in fluid consumption and taste discrimination.

Decreased Ethanol Preference and Consumption in Dopamine Transporter Female Knock‐Out Mice

It is commonly believed that the mesolimbic dopamine (DA) system participates in the etiology of alcoholism. One of the most important regulators of DA synaptic transmission is the DA transporter (DAT). We examined the effects of the genetic reduction or deletion of DAT on voluntary ethanol consumption and ethanol-induced sedation. Ethanol preference and consumption were assessed in the two-bottle choice paradigm, and the depressant effects of ethanol were evaluated by measuring sleep time after acute injection of ethanol. The latent period to lose the righting reflex was shorter in both knock-out mice (DAT-KO) and heterozygote mice (HET) than in wild-type (WT) mice. No significant difference was found among the three genotypes in the ethanol blood concentration at the onset of regaining the righting reflex. Females of all genotypes consumed more fluid than males of the same genotype. HET and DAT-KO females had increased total fluid consumption compared with WT females. DAT-KO males had increased fluid consumption compared with WT and HET males. Ethanol preference and consumption were not different among male mice of different genotypes. WT and HET females demonstrated significantly higher ethanol consumption than males. HET female mice did not differ from WT mice in ethanol preference. There was no difference between HET and WT mice in the preference for saccharin or quinine solutions. DAT-KO females avoided ethanol, and their consumption and preference were lower than in WT and HET females, despite markedly increased total intake. DAT-KO mice also demonstrated altered taste preference for saccharin and quinine. Partial deletion of DAT results in increased fluid consumption in female mice but does not change ethanol preference in either sex. Complete deletion of DAT reduces ethanol preference in female mice; this may be due to a combination of the pharmacological actions of DAT deletion and alterations in fluid consumption and taste discrimination.

Anterior Pituitary Hypoplasia and Dwarfism in Mice Lacking the Dopamine Transporter

Deletion of the dopamine transporter (DAT) results in increased dopaminergic tone, anterior pituitary hypoplasia, dwarfism, and an inability to lactate. DAT elimination alters the spatial distribution and dramatically reduces the numbers of lactotrophs and somatotrophs in the pituitary. Despite having normal circulating levels of growth hormone and prolactin in blood, hypoplastic glands from DAT −/− mice fail to respond to secretagog stimulation. The effects of DAT deletion on pituitary function result from elevated DA levels that down-regulate the lactotroph D 2 DA receptors and depress hypothalamic growth hormone-releasing hormone content. These results reveal an unexpected and important role or DA in the control of developmental events in the pituitary gland and assign a critical role for hypothalamic DA reuptake in regulating these events.