Dopamine Transporter Knockout Mice Research Articles

Enhanced cognitive flexibility and phasic striatal dopamine dynamics in a mouse model of low striatal tonic dopamine.

The catecholamine neuromodulators dopamine and norepinephrine are implicated in motor function, motivation, and cognition. Although roles for striatal dopamine in these aspects of behavior are well established, the specific roles for cortical catecholamines in regulating striatal dopamine dynamics and behavior are less clear. We recently showed that elevating cortical dopamine but not norepinephrine suppresses hyperactivity in dopamine transporter knockout (DAT-KO) mice, which have elevated striatal dopamine levels. In contrast, norepinephrine transporter knockout (NET-KO) mice have a phenotype distinct from DAT-KO mice, as they show elevated extracellular cortical catecholamines but reduced baseline striatal dopamine levels. Here we evaluated the consequences of altered catecholamine levels in NET-KO mice on cognitive flexibility and striatal dopamine dynamics. In a probabilistic reversal learning task, NET-KO mice showed enhanced reversal learning, which was consistent with larger phasic dopamine transients (dLight) in the dorsomedial striatum (DMS) during reward delivery and reward omission, compared to WT controls. Selective depletion of dorsal medial prefrontal cortex (mPFC) norepinephrine in WT mice did not alter performance on the reversal learning task but reduced nestlet shredding. Surprisingly, NET-KO mice did not show altered breakpoints in a progressive ratio task, suggesting intact food motivation. Collectively, these studies show novel roles of cortical catecholamines in the regulation of tonic and phasic striatal dopamine dynamics and cognitive flexibility, updating our current views on dopamine regulation and informing future therapeutic strategies to counter multiple psychiatric disorders.

Evaluation and Validation of Commercially Available Dopamine Transporter Antibodies

With a wide variety of dopamine transporter (DAT) antibodies available commercially, it is important to validate which antibodies provide sufficient immunodetection for reproducibility purpose and for accurate analysis of DAT levels and/or location. Commercially available DAT antibodies that are commonly used were tested in western blotting (WB) on wild-type (WT) and DAT-knock-out (DAT-KO) brain tissue and with immunohistology (IH) techniques against coronal slices of unilaterally lesioned 6-OHDA rats, in addition to wild-type and DAT-knock-out mice. DAT-KO mice and unilateral 6-OHDA lesions in rats were used as a negative control for DAT antibody specificity. Antibodies were tested at various concentrations and rated based on signal detection varying from no signal to optimal signal detection. Commonly used antibodies, including AB2231 and PT-22 524-1-AP, did not provide specific DAT signals in WB and IH. Although certain antibodies provided a good DAT signal, such as SC-32258, D6944, and MA5-24796, they also presented nonspecific bands in WB. Many DAT antibodies did not detect the DAT as advertised, and this characterization of DAT antibodies may provide a guide for immunodetection of DAT for molecular studies.

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.

Cannabidiol, but Not Δ9-Tetrahydrocannabinol, Has Strain- and Genotype-Specific Effects in Models of Psychosis.

Introduction: Cannabis use has been associated with an increased incidence of psychiatric disorders, yet the underlying neurobiological processes mediating these associations are poorly understood. Whereas exposure to Δ9-tetrahydrocannabinol (THC) has been associated with the development or exacerbation of psychosis, treatment with cannabidiol (CBD) has been associated with amelioration of psychosis. In this study, we demonstrate a complex effect of CBD in mouse models of psychosis, based on factors, including dose, strain, and genotype. Methods: Adult GluN1 knockdown (GluN1KD) and dopamine transporter knockout (DATKO) mice (almost equally balanced for male/female) were acutely treated with vehicle, THC (4 mg/kg), CBD (60, 120 mg/kg), or THC:CBD (1:15, 4:60 mg/kg) and tested in behavioral assays. Results: GluN1KD and DATKO mice displayed hyperactivity, impaired habituation, and sensorimotor gating, along with increased stereotypy and vertical activity. THC, alone and in combination with CBD, produced a robust "dampening" effect on the exploratory behavior regardless of strain or genotype. CBD exhibited a more complex profile. At 60 mg/kg, CBD had minimal effects on horizontal activity, but the effects varied in terms of directionality (increase vs. decrease) in other parameters; effects on stereotypic behaviors differ by genotype, while effects on vertical exploration differ by strain×genotype. CBD at 120 mg/kg had a "dampening" effect on exploration overall, except in GluN1KD mice, where no effect was observed. In terms of sensorimotor gating, both THC and CBD had minimal effects, except for 120 mg/kg CBD, which exacerbated the acoustic startle response. Conclusions: Here, we present a study that highlights the complex mechanism of phytocannabinoids, particularly CBD, in models of psychosis-like behavior. These data require careful interpretation, as agonism of the cannabinoid receptor 1 (CB1) resulting in a decrease in locomotion can be misinterpreted as "antipsychotic-like" activity in murine behavioral outputs of psychosis. Importantly, the THC-mediated decrease in hyperexploratory behavior observed in our models (alone or in combination) was not specific to the genetic mutants, but rather was observed regardless of strain or genotype. Furthermore, CBD treatment, when comparing mutants with their wild-type littermate controls, showed little to no "antipsychotic-like" activity in our models. Therefore, it is not only important to consider dose when designing/interpreting therapeutically driven phytocannabinoid studies, but also effects of strain or genetic vulnerability respective to the general population.

0756 Preclinical Pharmacology of Solriamfetol: Potential Mechanisms for Wake Promotion

Abstract Introduction Solriamfetol is a wake-promoting agent (WPA) approved for the treatment of excessive daytime sleepiness associated with narcolepsy and obstructive sleep apnea. The wake-promoting mechanism of solriamfetol may result from dopamine and norepinephrine reuptake inhibition. Preclinical pharmacology studies were conducted to further elucidate the molecular targets activated by solriamfetol and compare them to that of known WPAs and traditional stimulants. Methods In vitro binding and functional studies were conducted in a panel of cell lines or membrane preparations expressing transmembrane receptors and monoamine transporters to measure the activity of solriamfetol, comparator WPAs, and traditional stimulants. Electrophysiology studies were conducted in slice preparations from mouse ventral tegmental area (VTA). Studies to measure locomotor activity and wake-promoting effects were conducted in mice. Results In vitro functional studies showed agonist activity of solriamfetol at human, mouse, and rat TAAR1 receptors. hTAAR1 EC50 values (10–16 µM) were within the clinically observed therapeutic solriamfetol plasma concentration range and overlapped with the observed DAT/NET inhibitory potencies of solriamfetol in vitro. TAAR1 agonist activity was unique to solriamfetol; neither the WPA modafinil nor the DAT/NET inhibitor bupropion had TAAR1 agonist activity. Solriamfetol (1–10 µM) dose-dependently inhibited the firing frequency of dopaminergic VTA neurons in mouse brain slices, similar to known TAAR1 agonists; however, these effects were inhibited by a D2 antagonist, suggesting a DAT-mediated effect. Unlike traditional stimulants, solriamfetol did not increase locomotor activity in naive mice, but inhibited the increase in locomotor activity in DAT knockout mice. Conclusion Preclinical studies have identified agonist activity at the TAAR1 receptor and, possibly, lower potency agonist activity at 5-HT1A receptors as potential pharmacological targets for solriamfetol, in addition to its activity as a DAT/NET inhibitor. Given the current understanding of TAAR1 agonists as modulators of monoamine transmission with potential wake-promoting effects in multiple preclinical species, agonist activity at the hTAAR1 receptor may represent an additional pharmacological target underlying the wake-promoting effects for solriamfetol, in addition to its DNRI activity. Support (If Any) Jazz Pharmaceuticals

A Role for Cortical Dopamine in the Paradoxical Calming Effects of Psychostimulants

Attention deficit hyperactivity disorder (ADHD) affects children and adults, and manifests symptoms such as hyperactivity, impulsivity and cognitive disabilities. Psychostimulants, which are the primary treatment for ADHD, target monoamine transporters and have a paradoxical calming effect, but their mechanism of action is unclear. Studies using the dopamine (DA) transporter (DAT) knockout mice, which have elevated striatal DA levels and are considered an animal model of ADHD, have suggested that the paradoxical calming effect of psychostimulants might be through the actions on serotonin neurotransmission. On the other hand, newer non‐stimulant class of drugs such as atomoxetine and guanfacine suggest that targeting the norepinephrine (NE) system in the PFC might explain this paradoxical calming effect. We sought to decipher the mechanism of this paradoxical effect of psychostimulants through an integrated approach using ex vivo monoamine efflux experiments, monoamine transporter knockout mice, drug infusions and behavior. Our ex vivo efflux experiments reveal that NE transporter (NET) blocker desipramine elevates both norepinephrine and dopamine but not serotonin levels, in PFC tissue slices from wild‐type and DAT‐KO but not NET‐KO mice. However, serotonin (5‐HT) transporter (SERT) inhibitor fluoxetine elevates only serotonin in all three genotypes. Systemic administration of both desipramine and fluoxetine but local PFC infusion of only desipramine and not fluoxetine inhibits hyperactivity in the DAT‐KO mice. In contrast, pharmacological norepinephrine depletion but dopamine elevation using Nepicastat also inhibits hyperactivity in DAT‐KO mice. Together, these data suggest that elevation of PFC dopamine and not norepinephrine or serotonin as a convergent mechanism for the paradoxical psychostimulant effects observed in ADHD therapy.

Effectiveness and Relationship between Biased and Unbiased Measures of Dopamine Release and Clearance.

Fast-scan cyclic voltammetry (FSCV) is an effective tool for measuring dopamine release and clearance throughout the brain, especially the striatum where dopamine terminals are abundant and signals are heavily regulated by release machinery and the dopamine transporter (DAT). Peak height measurement is perhaps the most common method for measuring dopamine release, but it is influenced by changes in clearance. Michaelis-Menten-based modeling has been a standard in measuring dopamine clearance, but it is problematic in that it requires experimenter fitted modeling subject to experimenter bias. This study presents the use of the first derivative (velocity) of evoked dopamine signals as an alternative approach for measuring and distinguishing dopamine release from clearance. Maximal upward velocity predicts reductions in dopamine peak height due to D2 and GABAB receptor stimulation and by alterations in calcium concentrations. The Michaelis-Menten maximal velocity (Vmax) measure, an approximation for DAT levels, predicts maximal downward velocity in slices and in vivo. Dopamine peak height and upward velocity were similar between wild-type and DAT knock-out (DATKO) mice. In contrast, downward velocity was lower and exponential decay (tau) was higher in DATKO mice, supporting the use of both measures for extreme changes in DAT activity. In slices, the competitive DAT inhibitors cocaine, PTT, and WF23 increased peak height and upward velocity differentially across increasing concentrations, with PTT and cocaine reducing these measures at high concentrations. Downward velocity and tau values decreased and increased respectively across concentrations, with greater potency and efficacy observed with WF23 and PTT. In vivo recordings demonstrated similar effects of WF23, PTT, and cocaine on measures of release and clearance. Tau was a more sensitive measure at low concentrations, supporting its use as a surrogate for the Michaelis-Menten measure of apparent affinity (Km). Together, these results inform on the use of these various measures for dopamine release and clearance.

A role for cortical dopamine in the paradoxical calming effects of psychostimulants

Psychostimulants have a paradoxical calming effect in the treatment of attention deficit hyperactivity disorder (ADHD), but their mechanism of action is unclear. Studies using dopamine (DA) transporter (DAT) knockout (KO) mice have suggested that the paradoxical calming effect of psychostimulants might occur through actions on serotonin (5-HT) neurotransmission. However, newer non-stimulant drugs, such as atomoxetine and guanfacine, suggest that targeting the norepinephrine (NE) system in the prefrontal cortex (PFC) might explain this paradoxical calming effect. Thus, we sought to clarify the mechanism of this paradoxical action of psychostimulants. Our ex vivo efflux experiments reveal that the NE transporter (NET) blocker desipramine elevates both norepinephrine (NE) and dopamine (DA), but not 5-HT levels, in PFC tissue slices from wild-type (WT) and DAT-KO, but not NET-KO mice. However, the 5-HT transporter (SERT) inhibitor fluoxetine elevates only 5-HT in all three genotypes. Systemic administration of desipramine or fluoxetine inhibits hyperactivity in DAT-KO mice, whereas local PFC infusion of desipramine alone produced this same effect. In contrast, pharmacological NE depletion and DA elevation using nepicastat also inhibits hyperactivity in DAT-KO mice. Together, these data suggest elevation of PFC DA and not NE or 5-HT, as a convergent mechanism for the paradoxical effects of psychostimulants observed in ADHD therapy.

Neurokinin-1 Antagonism Distinguishes the Role of Norepinephrine Transporter from Dopamine Transporter in Mediating Amphetamine Behaviors

Background: Amphetamine (AMPH) and other psychostimulants act on the norepinephrine (NE) transporter (NET) and the dopamine (DA) transporter (DAT) and enhance NE and DA signaling. Both NET and DAT share anatomical and functional characteristics and are regulated similarly by psychostimulants and receptor-linked signaling pathways. We and others have demonstrated that NET and DAT are downregulated by AMPH and substance P/neurokinin-1 receptor (NK1R)-mediated protein kinase C pathway. Objectives: Since both NET and DAT are downregulated by AMPH and NK1R activation and share high sequence homology, the objective of the study was to determine the catecholamine transporter specificity in NK1R modulation of AMPH-induced behaviors. Methods: The effect of NK1R antagonism on AMPH-induced conditioned place preference (CPP) as well as AMPH-induced NET and DAT downregulation was examined using NET and DAT knockout mice (NET-KO and DAT-KO) along with their wild-type littermates. Results: Aprepitant (5 mg/kg i.p.) significantly attenuated AMPH (2 mg/kg i.p.)-induced CPP in the wild-type and DAT-KO but not in the NET-KO. Locomotor activity measured during the post-conditioning test (in the absence of AMPH) showed higher locomotor activity in DAT-KO compared to wild-type or NET-KO. However, the locomotor activity of all 3 genotypes remained unchanged following aprepitant. Additionally, in the ventral striatum of wild-type, the AMPH-induced downregulation of NET function and surface expression but not that of DAT was attenuated by aprepitant. Conclusions: The results from the current study demonstrate that aprepitant attenuates the expression of AMPH-induced CPP in DAT-KO mice but not in NET-KO mice suggesting a role for NK1R-mediated NET regulation in AMPH-induced behaviors.

Enhanced Dopamine Transmission and Hyperactivity in the Dopamine Transporter Heterozygous Mice Lacking the D3 Dopamine Receptor.

Dopamine transporter knockout (DATk) mice are known to demonstrate profound hyperactivity concurrent with elevated (5-fold) extracellular dopamine in the basal ganglia. At the same time, heterozygous DAT mice (DATh) demonstrate a 2-fold increase in dopamine levels yet only a marginal elevation in locomotor activity level. Another model of dopaminergic hyperactivity is the D3 dopamine receptor knockout (D3k) mice, which present only a modest hyperactivity phenotype, predominately manifested as stereotypical behaviors. In the D3k mice, the hyperactivity is also correlated with elevated extracellular dopamine levels (2-fold) in the basal ganglia. Cross-breeding was used to evaluate the functional consequences of the deletion of both genes. In the heterozygous DAT mice, inactivation of the D3R gene (DATh/D3k) resulted in significant hyperactivity and further elevation of striatal extracellular dopamine above levels observed in respective single mutant mice. The decreased weight of DATk mice was evident regardless of the D3 dopamine receptor genotype. In contrast, measures of thermoregulation revealed that the marked hypothermia of DATk mice (−2 °C) was reversed in double knockout mice. Thus, the extracellular dopamine levels elevated by prolonging uptake could be elevated even further by eliminating the D3 receptor. These data also suggest that the hypothermia observed in DATk mice may be mediated through D3 receptors.

A novel allosteric modulator of the cannabinoid CB1 receptor ameliorates hyperdopaminergia endophenotypes in rodent models

The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB1 receptor (CB1R) allosteric modulator, ABM300, to ameliorate these dysregulated behaviors. ABM300 was characterized in vitro (receptor binding, β-arrestin2 recruitment, ERK1/2 phosphorylation, cAMP inhibition) and in vivo (anxiety-like behaviors, cannabimimetic effects, novel environment exploratory behavior, pre-pulse inhibition, conditioned avoidance response) to assess the effects of the compound in dysregulated behaviors within the transgenic models. In vitro, ABM300 increased CB1R agonist binding but acted as an inhibitor of CB1R agonist induced signaling, including β-arrestin2 translocation, ERK phosphorylation and cAMP inhibition. In vivo, ABM300 did not elicit anxiogenic-like or cannabimimetic effects, but it decreased novelty-induced hyperactivity, exaggerated stereotypy, and vertical exploration in both transgenic models of hyperdopaminergia, as well as normalizing PPI in DATKO mice. The data demonstrate for the first time that a CB1R allosteric modulator ameliorates the behavioral deficits in two models of increased dopamine, warranting further investigation as a potential therapeutic target in psychiatry.

Potential role of primed microglia during obesity on the mesocorticolimbic circuit in autism spectrum disorder.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disease which involves functional and structural defects in selective central nervous system (CNS) regions that harm function and individual ability to process and respond to external stimuli. Individuals with ASD spend less time engaging in social interaction compared to non-affected subjects. Studies employing structural and functional magnetic resonance imaging reported morphological and functional abnormalities in the connectivity of the mesocorticolimbic reward pathway between the nucleus accumbens and the ventral tegmental area (VTA) in response to social stimuli, as well as diminished medial prefrontal cortex in response to visual cues, whereas stronger reward system responses for the non-social realm (e.g., video games) than social rewards (e.g., approval), associated with caudate nucleus responsiveness in ASD children. Defects in the mesocorticolimbic reward pathway have been modulated in transgenic murine models using D2 dopamine receptor heterozygous (D2+/-) or dopamine transporter knockout mice, which exhibit sociability deficits and repetitive behaviors observed in ASD phenotypes. Notably, the mesocorticolimbic reward pathway is modulated by systemic and central inflammation, such as primed microglia, which occurs during obesity or maternal overnutrition. Therefore, we propose that a positive energy balance during obesity/maternal overnutrition coordinates a systemic and central inflammatory crosstalk that modulates the dopaminergic neurotransmission in selective brain areas of the mesocorticolimbic reward pathway. Here, we will describe how obesity/maternal overnutrition may prime microglia, causing abnormalities in dopamine neurotransmission of the mesocorticolimbic reward pathway, postulating a possible immune role in the development of ASD.

Regional Effects of Monoamine Transporter Blockers on Extracellular Monoamine Levels

Monoamine transporter inhibitors are used in the treatment of a number of psychiatric disorders, including major depression and ADHD, and have high abuse potential. These drugs increase extracellular levels of monoamines by blocking the reuptake of monoamines through their respective transporters. Although selective norepinephrine (NE), dopamine (DA) and serotonin (5‐HT) transporter inhibitors increase NE, DA and 5‐HT, respectively, through blockade of their cognate transporter, there is substantial evidence showing that these selective inhibitors can also block the reuptake of monoamines from non‐cognate transporters. For example, studies have shown that norepinephrine transporter (NET) inhibitors can increase extracellular NE and DA levels in the PFC and hippocampus. Studies have also shown increased extracellular NE, DA and 5‐HT levels in the PFC by administration of a selective serotonin reuptake inhibitor (SSRI). Moreover, studies from dopamine transporter (DAT) and serotonin transporter (SERT) KO mice have also shown that selective NET and SERT blockers can increase extracellular DA levels in both the PFC and striatum. Although these non‐selective effects have been reported, there has not been a systematic investigation of brain‐region specific effects of selective monoamine transporter inhibitors on extracellular monoamine levels. We therefore sought to determine the effects of selective inhibitors of monoamine transporters on extracellular monoamine levels in the prefrontal cortex (PFC), nucleus accumbens (NAcc) and dorsal striatum (dStr). We used an ex vivo neurotransmitter release assay to determine the dose‐dependent effects of a NET inhibitor, desipramine, a DAT inhibitor, GRB 12909 and a SERT inhibitor, fluoxetine, on extracellular NE, DA and 5‐HT from PFC, NAcc, and dStr slices of wild‐type (WT) mice. We also examined the dose‐dependent effects of amphetamine on extracellular monoamine levels in the brain regions of interest of WT mice. The findings from WT mice were subsequently compared to those obtained from DAT and NET knockout mice. The results show that selective blockade of NET, DAT and SERT increase extracellular levels of NE, DA, and 5‐HT respectively, as well as alter the extracellular levels of non‐selective monoamines in a brain‐region and genotype specific manner. These findings will guide future studies exploring how region‐specific changes in monoamine levels affect the activity of neuronal circuits, physiology and behavior.Support or Funding InformationBBRF/NARSAD Young Investigator award to Nikhil Urs

Role of Cortical Catecholamines in the Paradoxical Action of Psychostimulants

Attention deficit hyperactivity disorder (ADHD) affects young children and manifests symptoms such as hyperactivity, impulsivity and cognitive disabilities. The primary line of treatment for ADHD are the psychostimulant class of drugs including amphetamine (Adderall) and methylphenidate (Ritalin). Several theories have been postulated on the paradoxical calming effect of psychostimulants on ADHD patients, but even after decades of research, their exact mechanism of action is unknown. Clinical studies have shown reduced levels of dopamine (DA) receptor and DA transporters (DAT) in ADHD patients, presumably due to elevated caudate DA levels. Studies using the dopamine (DA) transporter (DAT) knockout mice, which have elevated striatal DA levels and are considered an animal model of ADHD, have suggested that the paradoxical calming effect of psychostimulants might be through the parallel actions of other monoamine neurotransmitters. On the other hand, newer non‐stimulant classes of drugs such as atomoxetine and Intuniv suggest that targeting the norepinephrine (NE) system might explain the paradoxical calming effect. Again, the molecular and cellular mechanisms of action for these drugs or neurotransmitters in ADHD therapy is unknown.Previous work has shown that DA levels in the PFC have a reciprocal effect on striatal DA levels and locomotion. Our preliminary data suggest that PFC D2+ pyramidal neurons project to the striatum and midbrain dopamine neurons and that infusion of D2 agonists in the PFC inhibits locomotion. We hypothesize that PFC DA through the action of PFC D2+ pyramidal neurons regulates striatal DA release and locomotion. To test our hypothesis we use ex vivo neurotransmitter release from mouse PFC slices, microdialysis and locomotor measurements, in NET and DAT knockout mice. Our preliminary data suggest that NET and SERT blockers not only increase extracellular levels of NE and 5HT, but also DA. These data suggest a possible role for cortical catecholamines in regulating the paradoxical effects of psychostimulants. Our future studies will not only shed light on the mechanism of psychostimulant action in ADHD therapy, but will facilitate development of newer therapies based on mechanistic evidence.Support or Funding InformationBrain Behavior Research Foundation 27765 to N. UrsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

No effect of sex on ethanol intake and preference after dopamine transporter (DAT) knockdown in adult mice.

Dopamine levels are controlled in part by transport across the cell membrane by the dopamine transporter (DAT), and recent evidence showed that a polymorphism in the gene encoding DAT is associated with alcoholism. However, research in animal models using DAT knockout mice has yielded conflicting results. The present study was planned to evaluate the effects of DAT knockdown in the nucleus accumbens (Nacc) on voluntary ethanol consumption and preference in male and female C57BL/6J mice. For this purpose, animals were stereotaxically injected with DAT siRNA-expressing lentiviral vectors in the Nacc, and using a voluntary, continuous access two-bottle choice model of alcohol, we investigated the importance of accumbal DAT expression in voluntary alcohol intake and preference. We also investigated the effects of DAT knockdown on saccharin and quinine consumption and ethanol metabolism. We show that females consumed more alcohol than males. Interestingly, DAT knockdown in the Nacc significantly decreased alcohol intake and preference in both groups, but no significant sex by group interaction was observed. Also, DAT knockdown did not alter total fluid consumption, saccharin or quinine consumption, or blood ethanol concentrations. Using Pearson correlation, results indicated a strong positive relationship between DAT mRNA expression and ethanol consumption and preference. Taken together, these data provide further evidence that DAT plays an important role in controlling ethanol intake and that accumbal DAT contributes in the modulation of the reinforcing effects of ethanol. Overall, the results suggest that DAT inhibitors may be valuable in the pharmacotherapy of alcoholism.

The high efficacy of muscarinic M4 receptor in D1 medium spiny neurons reverses striatal hyperdopaminergia

The opposing action of dopamine and acetylcholine has long been known to play an important role in basal ganglia physiology. However, the quantitative analysis of dopamine and acetylcholine signal interaction has been difficult to perform in the native context because the striatum comprises mainly two subtypes of medium-sized spiny neurons (MSNs) on which these neuromodulators exert different actions. We used biosensor imaging in live brain slices of dorsomedial striatum to monitor changes in intracellular cAMP at the level of individual MSNs. We observed that the muscarinic agonist oxotremorine decreases cAMP selectively in the MSN subpopulation that also expresses D1 dopamine receptors, an action mediated by the M4 muscarinic receptor. This receptor has a high efficacy on cAMP signaling and can shut down the positive cAMP response induced by dopamine, at acetylcholine concentrations which are consistent with physiological levels. This supports our prediction based on theoretical modeling that acetylcholine could exert a tonic inhibition on striatal cAMP signaling, thus supporting the possibility that a pause in acetylcholine release is required for phasic dopamine to transduce a cAMP signal in D1 MSNs. In vivo experiments with acetylcholinesterase inhibitors donepezil and tacrine, as well as with the positive allosteric modulators of M4 receptor VU0152100 and VU0010010 show that this effect is sufficient to reverse the increased locomotor activity of DAT-knockout mice. This suggests that M4 receptors could be a novel therapeutic target to treat hyperactivity disorders.

Reward-enhancing effect of methylphenidate is abolished in dopamine transporter knockout mice: A model of attention-deficit/hyperactivity disorder.

AimAttention‐deficit/hyperactivity disorder is a heterogeneous neurobiological disorder that is characterized by inattention, impulsivity, and an increase in motor activity. Although methylphenidate has been used as a medication for decades, unknown is whether methylphenidate treatment can cause drug dependence in patients with attention‐deficit/hyperactivity disorder. This study investigated the reward‐enhancing effects of methylphenidate using intracranial self‐stimulation in an animal model of attention‐deficit/hyperactivity disorder, dopamine transporter knockout mice.MethodsFor the intracranial self‐stimulation procedures, the mice were trained to nosepoke to receive direct electrical stimulation via an electrode that was implanted in the lateral hypothalamus. After the acquisition of nosepoke responding for intracranial self‐stimulation, the effects of methylphenidate on intracranial self‐stimulation were investigated.ResultsIn the progressive‐ratio procedure, dopamine transporter knockout mice exhibited an increase in intracranial self‐stimulation compared with wild‐type mice. Treatment with 5 and 10 mg/kg methylphenidate increased intracranial self‐stimulation responding in wild‐type mice. Methylphenidate at the same doses did not affect intracranial self‐stimulation responding in dopamine transporter knockout mice. We then investigated the effects of high‐dose methylphenidate (60 mg/kg) in a rate‐frequency procedure. High‐dose methylphenidate significantly decreased intracranial self‐stimulation responding in both wild‐type and dopamine transporter knockout mice.ConclusionsThese results suggest that low‐dose methylphenidate alters the reward system (ie, increases intracranial self‐stimulation responding) in wild‐type mice via dopamine transporter inhibition, whereas dopamine transporter knockout mice do not exhibit such alterations. High‐dose methylphenidate appears to suppress intracranial self‐stimulation responding not through dopamine transporter inhibition but rather through other mechanisms. These results support the possibility that methylphenidate treatment for attention‐deficit/hyperactivity disorder does not increase the risk of drug dependence, in attention‐deficit/hyperactivity disorder patients with dopamine transporter dysfunction.

Behavioral Phenotyping of Dopamine Transporter Knockout Rats: Compulsive Traits, Motor Stereotypies, and Anhedonia.

Alterations in dopamine neurotransmission are generally associated with diseases such as attention-deficit/hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD). Such diseases typically feature poor decision making and lack of control on executive functions and have been studied through the years using many animal models. Dopamine transporter (DAT) knockout (KO) and heterozygous (HET) mice, in particular, have been widely used to study ADHD. Recently, a strain of DAT KO rats has been developed (1). Here, we provide a phenotypic characterization of reward sensitivity and compulsive choice by adult rats born from DAT–HET dams bred with DAT–HET males, in order to further validate DAT KO rats as an animal model for preclinical research. We first tested DAT KO rats’ sensitivity to rewarding stimuli, provided by highly appetitive food or sweet water; then, we tested their choice behavior with an Intolerance-to-Delay Task (IDT). During these tests, DAT KO rats appeared less sensitive to rewarding stimuli than wild-type (WT) and HET rats: they also showed a prominent hyperactive behavior with a rigid choice pattern and a wide number of compulsive stereotypies. Moreover, during the IDT, we tested the effects of amphetamine (AMPH) and RO-5203648, a trace amine-associated receptor 1 (TAAR1) partial agonist. AMPH accentuated impulsive behaviors in WT and HET rats, while it had no effect in DAT KO rats. Finally, we measured the levels of tyrosine hydroxylase, dopamine receptor 2 (D2), serotonin transporter, and TAAR1 mRNA transcripts in samples of ventral striatum, finding no significant differences between WT and KO genotypes. Throughout this study, DAT KO rats showed alterations in decision-making processes and in motivational states, as well as prominent motor and oral stereotypies: more studies are warranted to fully characterize and efficiently use them in preclinical research.

Reinforcing Doses of Intravenous Cocaine Produce Only Modest Dopamine Uptake Inhibition.

The reinforcing efficacy of cocaine is thought to stem from inhibition of the dopamine transporter (DAT) and subsequent increases in extracellular dopamine concentrations in the brain. In humans, this hypothesis has generally been supported by positron emission tomography imaging studies where the percent of DATs occupied by cocaine is used as a measure of cocaine activity in the brain. Interpretation of these studies, however, often relies on the assumption that measures of DAT occupancy directly correspond with functional DAT blockade. In the current studies, we used in vivo and in vitro fast scan cyclic voltammetry in mice to measure dopamine uptake inhibition following varying doses of cocaine as well as two high affinity DAT inhibitors. We then compared dopamine clearance rates following these drug treatments to dopamine clearance obtained from DAT knockout mice as a proxy for complete DAT blockade. We found that administration of abused doses of cocaine resulted in approximately 2% of maximal DAT blockade. Overall, our data indicate that abused doses of cocaine produce a relatively modest degree of DA uptake inhibition, and suggest that the relationship between DAT occupancy and functional blockade of the DAT is more complex than originally posited.

Distinct Roles of Opioid and Dopamine Systems in Lateral Hypothalamic Intracranial Self-Stimulation.

Background:Opioid and dopamine systems play crucial roles in reward. Similarities and differences in the neural mechanisms of reward that are mediated by these 2 systems have remained largely unknown. Thus, in the present study, we investigated the differences in reward function in both µ-opioid receptor knockout mice and dopamine transporter knockout mice, important molecules in the opioid and dopamine systems.Methods:Mice were implanted with electrodes into the right lateral hypothalamus (l hour). Mice were then trained to put their muzzle into the hole in the head-dipping chamber for intracranial electrical stimulation, and the influences of gene knockout were assessed.Results:Significant differences are observed between opioid and dopamine systems in reward function. µ-Opioid receptor knockout mice exhibited enhanced intracranial electrical stimulation, which induced dopamine release. They also exhibited greater motility under conditions of “despair” in both the tail suspension test and water wheel test. In contrast, dopamine transporter knockout mice maintained intracranial electrical stimulation responding even when more active efforts were required to obtain the reward.Conclusions:The absence of µ-opioid receptor or dopamine transporter did not lead to the absence of intracranial electrical stimulation responsiveness but rather differentially altered it. The present results in µ-opioid receptor knockout mice are consistent with the suppressive involvement of µ-opioid receptors in both positive incentive motivation associated with intracranial electrical stimulation and negative incentive motivation associated with depressive states. In contrast, the results in dopamine transporter knockout mice are consistent with the involvement of dopamine transporters in positive incentive motivation, especially its persistence. Differences in intracranial electrical stimulation in µ-opioid receptor and dopamine transporter knockout mice underscore the multidimensional nature of reward.