Dopamine Transporter Knockout Mice Research Articles

Local Knockdown of Genes in the Brain Using Small Interfering RNA: A Phenotypic Comparison with Knockout Animals

Recent reports have suggested effectiveness of RNA interference (RNAi) for the analysis of gene functions in the brain. This study sought to determine the efficiency of local small interfering RNA (siRNA) injections, comparing this approach with animals generated through classical gene targeting. Small interfering RNA against dopamine transporter (DAT) (35 microg/14 days) or tyrosine hydroxylase (TH) (15 microg/3 days) was injected into the ventral tegmental/substantia nigra areas of the brain of adult wildtype or DAT-knockout mice, respectively. Local injections of siRNA resulted in a 35% to 40% reduction of DAT and TH protein levels in the striatum, respectively. Despite negligible effect of DAT knockdown on novelty-induced locomotion, the locomotor response of DAT siRNA treated animals to amphetamine was blunted similar to what is observed in the DAT heterozygote animals. Since incomplete reduction of TH levels in normal mice does not produce behavioral effects, TH siRNA experiments were carried out in DAT-knockout animals that show increased dependence on newly synthesized dopamine. Knockdown of TH in these animals resulted in reduced basal locomotion. Local injection of siRNA in the brain reduced gene expression by 40% to 50%, suggesting that siRNA-mediated knockdown of genes in the brain can be a complementary tool to classical transgenesis for the analysis of gene functions.

Norepinephrine Transporter Blockade can Normalize the Prepulse Inhibition Deficits Found in Dopamine Transporter Knockout Mice

Dopamine transporter knockout (DAT KO) mice display deficits in sensorimotor gating that are manifested by reduced prepulse inhibition (PPI) of the acoustic startle reflex. Since PPI deficits may model some of the cognitive dysfunctions identified in certain neuropsychiatric patients, we have studied the effects of transporter blockers on PPI in wild-type and DAT KO mice. Treatments with High dose psychostimulants that block DAT as well as the norepinephrine (NET) and serotonin (SERT) transporters (60 mg/kg cocaine or methylphenidate) significantly impaired PPI in wild-type mice. By contrast, these treatments significantly ameliorated the PPI deficits observed in untreated DAT KO mice. In studies with more selective transport inhibitors, the selective NET inhibitor nisoxetine (10 or 30 mg/kg) also significantly reversed PPI deficits in DAT KO mice. By contrast, while the SERT inhibitor fluoxetine (30 mg/kg) normalized these PPI deficits in DAT KO mice, citalopram (30 or 100 mg/kg) failed to do so. The 'paradoxical' effects of cocaine and methylphenidate in DAT KO mice are thus likely to be mediated, at least in part by the ability of these drugs to block NET, although serotonin systems may also have some role. Together with recent microdialysis data, these results support the hypothesis that prefrontal cortical NET blockade and consequent enhancement of prefrontal cortical extracellular dopamine mediates the reversal of PPI deficits in DAT KO mice.

No role of the dopamine transporter in acute ethanol effects on striatal dopamine dynamics

The acute effects of ethanol on dopamine (DA) release and clearance in the caudate-putamen were evaluated in wild-type and dopamine transporter (DAT) knockout (DAT-KO) mice, using microdialysis and voltammetry. Dialysate DA levels were elevated, approximately 80% above baseline levels, after administration of 2 g/kg ethanol in both wild-type and DAT-KO mice. In brain slices containing the caudate-putamen, a low (20 mM) concentration of ethanol produced no change in electrically stimulated DA release in either wild-type or DAT-KO mice. A high concentration (200 mM) of ethanol caused a similar decrease in DA release in slices from both types of mice. DA clearance was unaltered across the genotypes at low and high concentrations of ethanol. The fact that ethanol had similar effects in wild-type and DAT-KO mice, measured by in vivo microdialysis or brain slice voltammetry, supports the idea that acute ethanol does not interact with the DAT to produce its effects on the DA system.

Endocannabinoids Activate Transient Receptor Potential Vanilloid 1 Receptors to Reduce Hyperdopaminergia-Related Hyperactivity: Therapeutic Implications

Knockout (KO) mice invalidated for the dopamine transporter (DAT) constitute a powerful animal model of neurobiological alterations associated with hyperdopaminergia relevant to schizophrenia and attention-deficit/hyperactivity disorder (ADHD). Because of continuously increasing evidence for a neuromodulatory role of endocannabinoids in dopamine-related pathophysiological responses, we assessed endocannabinoid signaling in DAT KO mice and evaluated the ability of endocannabinoid ligands to normalize behavioral deficits, namely spontaneous hyperlocomotion in these mice. In DAT KO mice, we found markedly reduced anandamide levels, specifically in striatum, the dopamine nerve terminal region. Furthermore, three distinct indirect endocannabinoid agonists, the selective anandamide reuptake inhibitors AM404 and VDM11 and the fatty acid amidohydrolase inhibitor AA5HT, attenuated spontaneous hyperlocomotion in DAT KO mice. The hypolocomotor effects of AM404, VDM11, and AA5HT were significantly attenuated by co-administration of the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine but not the selective cannabinoid type 1 (CB1)receptor antagonist AM251. Interestingly, TRPV1 binding was increased in the striatum of DAT KO mice, while CB1 receptor binding was unaffected. These data indicate a dysregulated striatal endocannabinoid neurotransmission associated with hyperdopaminergic state. Restoring endocannabinoid homeostasis in active synapses might constitute an alternative therapeutic strategy for disorders associated with hyperdopaminergia. In this process, TRPV1 receptors seem to play a key role and represent a novel promising pharmacological target.

Manganese accumulation in striatum of mice exposed to toxic doses is dependent upon a functional dopamine transporter

The objective of this study was to determine the importance of the dopamine transporter (DAT) in manganese transport. Excessive manganese exposure is associated with a neurotoxicological disease known as manganism characterized by a specific accumulation of manganese in dopamine-rich brain regions. It has been hypothesized that the DAT mediates this specific transport, but its role in manganese neurotoxicity has not been directly examined. We examined brain tissues from manganese-exposed dopamine transporter knockout (DAT-KO) and wild-type (WT) mice. There was significantly less ( p < 0.05) manganese in the striatum of exposed DAT-KO mice compared to WT. However, the absence of a functioning DAT did not affect manganese accumulation in other brain regions examined. Furthermore, both iron and divalent metal transporter levels (two known modulators of brain manganese) were similar between DAT-KO and WT mice in all brain regions. These studies demonstrate that the DAT is involved in the facilitation of striatal manganese accumulation and that it may play a critical role in mediating manganese neurotoxicity.

Magnetic resonance imaging at microscopic resolution reveals subtle morphological changes in a mouse model of dopaminergic hyperfunction

Structural abnormalities of the basal ganglia have been documented in several neuropsychiatric conditions associated with dysregulation of the dopamine system. However, the histological nature underlying these changes is largely unknown. Using magnetic resonance imaging at microscopic resolution (MRI, 9.4 T with 43 μm isotropic spatial resolution) and stereological techniques, we have investigated the effect of increased dopamine neurotransmission on brain morphology in mice with elevated extracellular dopamine, the dopamine transporter knockout (DAT-KO) mice. We first demonstrate the usefulness of MRI at microscopic resolution for the accurate identification and measurement of volumes of specific subregions, accounting for less than 0.03% (0.16 mm 3) of the volume of a mouse brain. Furthermore, the MRI analysis reveals a significantly lower volume (−9%) of the anterior striatum of DAT-KO mice, while the volume of other dopamine-related structures such as the posterior striatum and the substantia nigra pars reticulata is unchanged in comparison to wild type littermates. Stereological analysis performed in the same brains reveals that one important structural factor accounting for this selective change in volume is a reduction of 18% in the absolute number of neuronal cell bodies. The feasibility of assessing accurately small morphological alterations in mouse models, where the molecular and histological pathologies can be easily compared in a controlled manner, provides a paradigm to examine the relevance of selective brain volumetric changes associated with a number of neuropathological conditions.

Characterization of conditioned place preference to cocaine in congenic dopamine transporter knockout female mice

The dopamine transporter (DAT) is thought to play a major role in the rewarding effects of cocaine. Therefore, it is surprising that cocaine reveals conditioned effects in DAT knockout (DAT-KO) mice. To examine these findings further, we obtained complete dose-effect curves for DAT-KO and DAT wild-type (DAT-WT) mice in a cocaine conditioned place preference (CPP) procedure. Congenic C57BL6 background female DAT-KO and DAT-WT mice were conditioned in a three-compartment place preference apparatus. Conditioning consisted of three 30-min sessions with cocaine (2.5, 5.0, 10.0, 20.0, or 40.0 mg/kg) and three 30-min sessions with saline. The distribution of time in each choice compartment was determined after each pair of conditioning sessions (one cocaine and one saline session). DAT-WT mice revealed CPP over a wide range of cocaine doses (5.0-40 mg/kg), whereas DAT-KO mice revealed CPP over a more restricted range of doses, with consistent CPP only occurring with 10 mg/kg of cocaine. CPP for cocaine develops in both DAT-KO and DAT-WT mice; however, the dose range at which CPP develops is much more restricted in DAT-KO mice than in DAT-WT mice. These observations corroborate the significant role of DAT inhibition in cocaine's conditioned effects.

Food‐Reinforced Operant Behavior in Dopamine Transporter Knockout Mice: Enhanced Resistance to Extinction

Dopamine (DA) plays roles in circuits that are important for brain reward and in striatal brain regions that are important for certain types of habit learning. These processes in wildtype, heterozygous, and homozygous dopamine transporter knockout (DAT-KO) mice, which were mildly food deprived and allowed to make nose-poke responses for food reinforcement, were studied. The mice were given 20-min sessions of daily (a) baseline exposure to the operant chambers, (b) acquisition of nose-poke responses in which responses were reinforced under a fixed ratio (FR5) schedule, (c) a progressive ratio schedule in which the number of responses required to obtain food was gradually increased, and (d) extinction of responses in which nose pokes were not followed by food. Neither heterozygous nor homozygous DAT-KO mice differed from their wildtype litter mates in the number of nose pokes displayed during baseline exposures to the chambers, the number of sessions required for acquisition, the number of responses under the FR5 schedule, or the number of responses under the progressive ratio schedule. Interestingly, however, in the five extinction sessions in which food was no longer delivered by nose poking, homozygous DAT-KO mice exerted significantly more responses than mice of either of the other two genotypes. These lines of evidence suggest a greater resistance of DAT-KO mice to the elimination of the response and support roles of dopaminergic systems in habit memory.

Molecular Mechanisms Underlying the Rewarding Effects of Cocaine

The initially surprising observation that cocaine retains its rewarding effects in dopamine transporter (DAT) knockout (KO) mice led our laboratory to examine the effects of deletion of other monoaminergic genes on cocaine reward. Our initial approach to this problem was to combine DAT KO mice with serotonin transporter (SERT) KO mice to make combined DAT/SERT KO mice. The combination of these knockouts eliminates cocaine reward as assessed in the conditioned place preference (CPP) paradigm. We have also identified evidence that, in the absence of DAT, there is greater participation in cocaine reward by serotonin (SERT) and norepinephrine (NET) transporters. Both NET and SERT blockers (nisoxetine and fluoxetine) produced significant CPPs in DAT KO mice, but not in wild-type (WT) mice. The striking elimination of cocaine CPP in combined DAT/SERT KO mice contrasts with effects that we have identified in combined NET/SERT knockout mice, which display increases in cocaine reward, and with recent reports that suggest that DAT/NET combined KOs retain substantial cocaine CPP. Overall, these studies indicate important requirements for several monoaminergic system genes to fully explain cocaine reward, in particular those expressed by dopamine and serotonin systems.

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.

MDMA "ecstasy" alters hyperactive and perseverative behaviors in dopamine transporter knockout mice.

Mice lacking the gene for the dopamine transporter (DAT) show a unique behavioral phenotype characterized by locomotor hyperactivity and repetitive circling in a novel environment. The hyperactivity of DAT (-/-) mice can be attenuated by psychostimulants and by serotonin uptake inhibitors, suggesting an important role for serotonin in the attenuation of locomotor hyperactivity in these mice. These studies characterized the effects of 3,4-methylenedioxy-N-methylamphetamine (MDMA), a serotonin releaser, on the amount and patterns of locomotor activity in DAT (+/+) and (-/-) mice. We compared the locomotor patterns produced by MDMA to those observed in DAT (-/-) mice, and examined whether MDMA altered the hyperactivity and perseverative locomotor patterns in DAT (-/-) mice. The effects of MDMA (10, 30 mg/kg) on locomotor activity in DAT (+/+) mice were measured for 90 min in a video tracker system to determine the optimal dose for subsequent studies in DAT (+/+) and (-/-) mice. The effects of 20 mg/kg MDMA on patterns of locomotor activity in DAT (+/+) and (-/-) mice were measured for 90 min. In DAT (+/+) mice, MDMA increased locomotor activity and induced repetitive straight movement patterns. In DAT (-/-) mice, however, MDMA (20 mg/kg) attenuated the characteristic locomotor hyperactivity seen in these mice. In contrast, MDMA potentiated the thigmotaxis and decreased entropy observed in the DAT (-/-) mice. The effects of MDMA observed here demonstrate that the different aspects of the abnormal locomotor behavior exhibited by DAT (-/-) mice can be independently manipulated by pharmacological treatments.

The Interaction of the γ-Aminobutyric Acid Transporter GAT-1 with the Neurotransmitter Is Selectively Impaired by Sulfhydryl Modification of a Conformationally Sensitive Cysteine Residue Engineered into Extracellular Loop IV

The (Na+ + Cl-)-coupled gamma-aminobutyric acid (GABA) transporter GAT-1 keeps synaptic levels of this neurotransmitter low and thereby enables efficient GABA-ergic transmission. Extracellular loops (III, IV, and V) have been shown to contain determinants for GABA selectivity and affinity. Here we analyze the role of extracellular loop IV in transport by cysteine scanning mutagenesis. Fourteen residues of this loop have been replaced by cysteine. GABA transport by eight of the fourteen mutants is markedly more sensitive to inhibition by membrane-impermeant methane thiosulfate reagents than wild-type. Mutant A364C has high activity and is potently inhibited by the sulfhydryl reagent. GABA transport by the A364C/C74A double mutant, where the only externally accessible cysteine residue of the wild-type has been replaced by alanine, is also highly sensitive to the sulfhydryl reagents. Maximal sensitivity is observed in the presence of the cosubstrates sodium and chloride. A marked protection is afforded by GABA, provided sodium is present. This protection is also observed at 4 degrees C. The non-transportable analogue SKF100330A also protects the double mutant against sulfhydryl modification in the presence of sodium but has the opposite effect in its absence. Electrophysiological analysis shows that upon sulfhydryl modification of this mutant, GABA can no longer induce transport currents. The voltage dependence of the transient currents indicates an increased apparent affinity for sodium. Moreover, GABA is unable to suppress the transient currents. Our results indicate that part of extracellular loop IV is conformationally sensitive, and its modification selectively abolishes the interaction of the transporter with GABA.

Aberrant responses in social interaction of dopamine transporter knockout mice

The dopamine (DA) transporter (DAT) controls the temporal and spatial resolution of dopaminergic neurotransmission. Disruption of the Dat1 gene in mice leads to increased extracellular DA concentrations and reduced expression of D1- and D2-like receptors in striatum. The mutants are hyperactive in the open field and they display deficits in learning and memory. In humans, dopaminergic dysfunction has been associated with a number of different psychiatric disorders and some of these conditions are accompanied by abnormal social responses. To determine whether social responses were also impaired in DAT knockout (KO) mice, behaviors of group- and isolation-housed animals were compared. All group-housed animals readily established hierarchies. However, the social organizations of the mutants were changed over time. Under both group- and isolation-housed conditions, mutants exhibited increased rates of reactivity and aggression following mild social contact. In isolation, exposure to a novel environment exacerbated these abnormal responses. Regardless of housing context, stereotyped and perseverative patterns of social responses were a common feature of the KO repertoire. In fact, many abnormal behaviors were due to the emergence and predominance of these inflexible behaviors. These data suggest that KO mice may serve as a useful animal model for understanding not only how DA dysfunction contributes to social abnormalities, but also how behavioral inflexibility distorts their social responses.

Differential psychostimulant-induced activation of neural circuits in dopamine transporter knockout and wild type mice

Dopamine (DA) is a neurotransmitter that has been implicated in a wide variety of psychiatric disorders that include attention deficit-hyperactivity disorder (ADHD), schizophrenia, and drug abuse. Recently, we have been working with a mouse in which the gene for the DA transporter (DAT) has been disrupted. This mouse is hyperactive in the open field, displays an inability to inhibit ongoing behaviors, and is deficient on learning and memory tasks. Psychostimulants such as amphetamine and methylphenidate attenuate the hyperlocomotion of the mutants, but stimulate activity of the wild type (WT) controls. The objective of the present study is to examine the neural basis for the differential responses to psychostimulants in these mice. WT and DAT knockout (KO) animals were given vehicle or methylphenidate, amphetamine, or cocaine and brain sections were immunostained for Fos. In WT mice, methylphenidate induced Fos-like immunoreactivity (Fos-LI) in the mesostriatal and mesolimbocortical DA pathways that included the anterior olfactory nucleus, frontal association cortex, orbitofrontal cortex, cingulate cortex, caudate-putamen, globus pallidus, claustrum, lateral septum, nucleus accumbens, basolateral and central nuclei of the amygdala, bed nucleus of stria terminalis, subthalamic nucleus, substantia nigra, ventral tegmental area, and dorsal raphe. Additional areas of activation included the granular dentate gyrus, Edinger-Westphal nucleus, and periaqueductal gray. While the mutants showed little response in most of these same areas, the anterior olfactory nucleus, caudal caudate-putamen, lateral septum, basolateral and central nuclei of the amygdala, and bed nucleus of stria terminalis were activated. Amphetamine and cocaine produced similar changes to that for methylphenidate, except these psychostimulants also induced Fos-LI in the nucleus accumbens of the KO animals. Since the DAT gene is disrupted in the KO mouse, these findings suggest that dopaminergic mechanisms may mediate the WT responses, whereas non-dopaminergic systems predominate in the mutant. In the mutants, it appears that limbic areas and non-dopaminergic transmitter systems within these brain regions may mediate responses to psychostimulants. Inasmuch as the KO mouse may represent a useful animal model for ADHD and because psychostimulants such as cocaine are reinforcing to these animals, our results may provide some useful insights into the neural mechanisms—other than DA—that may contribute to the symptoms of ADHD and/or drug abuse in human patients.

Motor behaviour deficits and their histopathological and functional correlates in the nigrostriatal system of dopamine transporter knockout mice

Chronic dysregulation of dopamine homeostasis has been shown to induce behavioural impairment in dopamine transporter knockout mutant mice arising from the dysfunction of the mesolimbic and hypothalamo-infundibular system. Here, we assessed whether there are also any motor consequences of a chronic and constitutive hyperdopaminergia in the nigrostriatal system in dopamine transporter knockout mutant mice. For this, we analysed motor performances using tests assessing balance, coordinated motor skills (rotarod, pole test), stride lengths and locomotor activity. Dopamine transporter knockout mutant mice were markedly hyperactive in the open field with central compartment avoidance, as previously shown. However, sensorimotor integration was also found to be altered in dopamine transporter knockout mutant mice which displayed a reduced fore- and hind-limb mean stride length, impaired motor coordination on the pole test and reduced rearings in the open field. Moreover, dopamine transporter knockout mutant mice showed a slower task acquisition on the rotarod. Six-week-old dopamine transporter knockout wild type mice having the same femur size as adult dopamine transporter knockout mutant mice ruled out a possible size-effect bias. Whilst there was no significant difference in the striatal volume, we found a slight but significant reduction in neuronal density in the striatum but not in the nucleus accumbens of dopamine transporter knockout mutant mice. There was a reduced binding in the striatum and nucleus accumbens of dopamine 1 receptors ([ 3H]SCH 23390) and dopamine 2 receptors ([ 3H]YM-09151-2). There was no significant difference in the number of dopaminergic neurons in the substantia nigra between dopamine transporter knockout mutant mice and dopamine transporter knockout wild type mice. These results suggest an impaired functioning of the nigrostriatal system in dopamine transporter knockout mutant hyperdopaminergic mice, as illustrated by motor and sensorimotor integration deficits, despite their apparent hyperactivity. These dysfunctions may arise from combined striatal cell loss and/or functional changes of dopaminergic neurotransmission.

Dopaminergic mouse mutants: Investigating the roles of the different dopamine receptor subtypes and the dopamine transporter

This chapter focuses on the dopamine receptor and dopamine transporter knockout mice that exist and the knowledge that is obtained using these mutant mice. Five dopamine receptors and one dopamine transporter are responsible for the binding, signaling, and reuptake of the neurotransmitter dopamine. The complexity and importance of the receptors and the transporter are well documented, although the mechanisms that regulate the dopaminergic signals are far from being completely understood. The dopaminergic system is involved in locomotion, motivated behaviors, cognition, learning and memory, endocrine regulation, the renal and cardiovascular systems as well as many other biological functions yet to be discovered. Drugs that activate or block the receptors are not specific enough to distinguish the function of each dopamine receptor subtype. Rather, the drugs available recognize various receptor subtypes with slightly different affinities, making it difficult to use the drugs in vivo and to assign the resulting phenotype to just one receptor subtype. There are knockout mice for each of the dopamine receptor subtypes as well as the dopamine transporter. These mice make it possible to study the function of each of the dopamine receptors in vivo and the phenotype of the mice in the absence of one or two of these receptors. The knockout mice have been particularly useful in beginning to understand how the five receptors act and interact with respect to drug addiction, neurodegenerative diseases, psychiatric diseases, and regulation of the endocrine system to name a few examples.

Cocaine mechanisms: enhanced cocaine, fluoxetine and nisoxetine place preferences following monoamine transporter deletions

Cocaine blocks uptake by neuronal plasma membrane transporters for dopamine, serotonin and norepinephrine, producing subjective effects in humans that are both euphoric/rewarding and also fearful, jittery and aversive. Mice with gene knockouts of each of these transporters display cocaine reward, manifest by cocaine place preferences that are at least as great as wildtype values. Norepinephrine and serotonin receptor knockouts even display enhanced cocaine reward. One explanation for these observations could be that cocaine produces aversive or anhedonic effects by serotonin or norepinephrine receptor blockade in wildtype mice that are removed in serotonin or norepinephrine receptor knockouts, increasing net cocaine reward. Adaptations to removing one transporter could also change the rewarding valence of blocking the remaining transporters. To test these ideas, drugs that block serotonin transporter (fluoxetine), norepinephrine transporter (nisoxetine) or all three transporters (cocaine) were examined in single- or multiple-transporter knockout mice. Fluoxetine and nisoxetine acquire rewarding properties in several knockouts that are not observed in wildtype mice. Adding serotonin transporter knockout to norepinephrine transporter knockouts dramatically potentiates cocaine reward. These and previous data provide evidence that serotonin and norepinephrine transporter blockade can contribute to the net rewarding valence of cocaine. They identify neuroadaptations that may help to explain the retention of cocaine reward by dopamine and serotonin transporter knockout mice. They are consistent with emerging hypotheses that actions at the three primary brain molecular targets for cocaine each provide distinct contributions to cocaine reward and cocaine aversion in wildtype mice, and that this balance changes in mice that develop without dopamine, norepinephrine or serotonin transporters.

Intravenous cocaine induced-activity and behavioural sensitization in norepinephrine-, but not dopamine-transporter knockout mice.

Previously, it was reported that both norepinephrine transporter (NET) and dopamine transporter (DAT) knockout (KO) mice were sensitive to the reinforcing effects of cocaine. However, assessing the locomotor-stimulant effects of cocaine in these subjects has proven difficult due to significant differences in their baseline activity compared to wild-type controls. The present studies were designed to clarify the role of NET and DAT in the stimulant effects of acute and repeated cocaine utilizing these knockout mice, and thereby assess the role of these substrates in the locomotor stimulant effects of cocaine. Mice were habituated to the test environment for sufficient time to ensure equal baselines at the time of cocaine administration. Mice then received cocaine (3-25 mg/kg) intravenously according to a within-session cumulative dose-response design. Cocaine dosing was repeated at 48-h intervals for four sessions to assess behavioural sensitization. NET-KO mice exhibited a reduced response to acute cocaine administration compared to wild-type (WT) controls. However, comparable sensitization developed in NET-KO and WT mice. The DAT-KO and DAT-heterozygote (HT) mice displayed no locomotor activation following either acute or repeated cocaine administration. These data suggest a role for the NET in the acute response to cocaine, but no involvement in sensitization to cocaine. In contrast, DAT appears to be necessary for both the acute locomotor response to cocaine and the subsequent development of sensitization. In addition to existing data concerning the reinforcing effects of cocaine in DAT-KO mice, these data suggest a dissociation between the reinforcing and locomotor stimulant effects of cocaine.

Dopamine transporter knock-out mice are hypersensitive to 3-nitropropionic acid-induced striatal damage.

Evidence suggests that dopamine is involved in the modulation of striatal excitotoxic processes. To further investigate this issue, we studied the effects of systemic 'low-dose' (total dose, 340 mg/kg in 7 days) 3-nitropropionic acid (3-NP) intoxication in dopamine transporter knock-out mice (DAT-/-) compared to wildtype (DAT+/+) mice. Systemic 'low-dose' 3-NP induced a significant impairment in a rotarod task only in DAT-/- mice. Histopathology also demonstrated a significant reduction of the striatal volume (-7%, P < 0.05), neuronal density (-12.5%, P < 0.001) and absolute number estimates of striatal neurons (-11.5%, P < 0.001) in DAT-/- compared to DAT+/+ mice, with increased glial activation, independent of the degree of succinate dehydrogenase inhibition. These findings strengthen the hypothesis for dopamine modulation of excitotoxicity within the nigrostriatal system.

Lack of Cocaine Effect on Dopamine Clearance in the Core and Shell of the Nucleus Accumbens of Dopamine Transporter Knock-Out Mice

Cocaine produces its reinforcing effects primarily by inhibiting the dopamine transporter (DAT) at the level of presynaptic terminals and increasing extracellular levels of dopamine (DA). Surprisingly, in mice genetically lacking the DAT, cocaine was still able to elevate DA in the nucleus accumbens (NAc). This finding is critically important for explaining the persistence of cocaine reinforcement in DAT knock-out (DAT-KO) mice. However, the mechanism by which cocaine elevates DA is unclear. Here, we tested the recently proposed hypothesis that in the absence of the DAT, the norepinephrine transporter (NET) could provide an alternative uptake site for DA clearance. If true, cocaine could elevate DA levels through its inhibition of the NET. In vitro voltammetry, a technique well suited for evaluating the effects of drugs on DA uptake, was used in the present study. We report that both cocaine and desipramine, a potent NET inhibitor, failed to change DA clearance or evoked release in the NAc of mutant mice. Additionally, fluoxetine, a serotonin transporter (SERT) inhibitor, also had no effect on these parameters. These data rule out the involvement of accumbal NET or SERT in the cocaine-induced increase in extracellular DA in DAT-KO mice. Moreover, the present findings suggest that in the DAT-KO mice, cocaine acts primarily outside the NAc to produce its effects.