Sustained N-methyl-d-aspartate receptor hypofunction remodels the dopamine system and impairs phasic signaling.

Chronic N-methyl-d-aspartate receptor (NMDAR) hypofunction has been proposed as a contributing factor to symptoms of schizophrenia. However, it is unclear how sustained NMDAR hypofunction throughout development affects other neurotransmitter systems that have been implicated in the disease. Dopamine neuron biochemistry and activity were examined to determine whether sustained NMDAR hypofunction causes a state of hyperdopaminergia. We report that a global, genetic reduction in NMDARs led to a remodeling of dopamine neurons, substantially affecting two key regulators of dopamine homeostasis, i.e., tyrosine hydroxylase and the dopamine transporter. In NR1 knockdown mice, dopamine synthesis and release were attenuated, and dopamine clearance was increased. Although these changes would have the effect of reducing dopamine transmission, we demonstrated that a state of hyperdopaminergia existed in these mice because dopamine D2 autoreceptors were desensitized. In support of this conclusion, NR1 knockdown dopamine neurons have higher tonic firing rates. Although the tonic firing rates are higher, phasic signaling is impaired, and dopamine overflow cannot be achieved with exogenous high-frequency stimulation that models phasic firing. Through the examination of several parameters of dopamine neurotransmission, we provide evidence that chronic NMDAR hypofunction leads to a state of elevated synaptic dopamine. Compensatory mechanisms to attenuate hyperdopaminergia also impact the ability to generate dopamine surges through phasic firing.

Voltammetric characterization of the effect of monoamine uptake inhibitors and releasers on dopamine and serotonin uptake in mouse caudate-putamen and substantia nigra slices.

Fast scan cyclic voltammetry is an electrochemical technique used to measure dynamics of transporter-mediated monoamine uptake in real time and provides a tool to evaluate the detailed effects of monoamine uptake inhibitors and releasers on dopamine and serotonin transporter function. We measured the effects of cocaine, methylphenidate, 2beta-propanoyl-3beta-(4tolyl) tropane (PTT), fluoxetine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), phentermine and fenfluramine on dopamine and serotonin uptake following electrically stimulated release in mouse caudate-putamen and substantia nigra pars reticulata slices. We determined rank orders of uptake inhibition effects based on two variables; increases in apparent K(m) for dopamine and serotonin uptake and inhibition constant (K(i)) values. For example, the rank order of uptake inhibition based on apparent K(m) values at the dopamine transporter was amphetamine>or=PTT>or=methylphenidate>>methamphetamine=phentermine=MDMA>cocaine>>fluoxetine=fenfluramine, and at the serotonin transporter was fluoxetine=methamphetamine=fenfluramine=MDMA > amphetamine=cocaine=PTT>or=methylphenidate>phentermine. Additionally, changes in electrically stimulated release were documented. This is the first study using voltammetry to measure the effects of a wide range of monoamine uptake inhibitors and releasers on dopamine and serotonin uptake in mouse brain slices. These studies also highlight methodological considerations for comparison of effects between heterogeneous brain regions.

Exocytotic release of dopamine in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.

The present study used voltammetry to ascertain whether electrically stimulated somatodendritic dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice was due to exocytosis or dopamine transporter reversal, as has been debated. The maximal concentration of electrically evoked dopamine release was similar between ventral tegmental area slices from dopamine transporter knockout and C57BL/6 mice. Dopamine transporter blockade (10 microM nomifensine) in slices from C57BL/6 mice inhibited dopamine uptake but did not alter peak evoked dopamine release. In addition, dopamine release and uptake kinetics in ventral tegmental area slices from dopamine transporter knockout mice were unaltered by the norepinephrine transporter inhibitor, desipramine (10 microM), or the serotonin transporter inhibitor, fluoxetine (10 microM). Furthermore, maximal dopamine release in ventral tegmental area slices from both C57BL/6 and dopamine transporter knockout mice was significantly decreased in response to Na(+) channel blockade by 1 microM tetrototoxin, removal of Ca(2+) from the perfusion media and neuronal vesicular monoamine transporter inhibition by RO-04-1284 (10 microM) or tetrabenazine (10 and 100 microM). Finally, the glutamate receptor antagonists AP-5 (50 and 100 microM) and CNQX (20 and 50 microM) had no effect on peak somatodendritic dopamine release in C57BL/6 mice. Overall, these data suggest that similar mechanisms, consistent with exocytosis, govern electrically evoked dopamine release in ventral tegmental area slices from C57BL/6 and dopamine transporter knockout mice.

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.

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.

Voltammetric assessment of dopamine clearance in the absence of the dopamine transporter: no contribution of other transporters in core or shell of nucleus accumbens.

Cocaine elevates dopamine (DA) in the nucleus accumbens (NAc) by blocking the uptake of DA through the DA transporter (DAT). It is commonly believed that the reinforcing properties of cocaine depend upon interaction with the DAT, however, cocaine is still reinforcing in mice with a genetic deletion of the DAT (DAT-KO mice). Although cocaine continues being able to elevate DA in the NAc of these mice, this mechanism is unclear. The present voltammetric study in brain slices was designed to examine the role of the norepinephrine and serotonin transporters in removing DA from the extracellular space in the NAc of DAT-KO mice. We found no effects of any monoamine uptake inhibitors, including cocaine (10 microM), desipramine (10 microM) or fluoxetine (10 microM) on the clearance of DA in these mice. Therefore, it appears that there is no compensatory uptake of DA by alternative transporters either in core or shell of the nucleus accumbens of DAT-KO mice.

Motivation on the Mediterranean: reward, compulsions and habit formation.

The 2007 Motivational Neuronal Networks meeting, held in Porquerolles, France was organized to generate debate and discussion on issues relating to reward, compulsion, and habit formation. The conference consisted primarily of four workshops that brought researchers from a wide variety of fields together in an informal atmosphere designed to facilitate interaction. This report is based on the detailed notes taken during the wide-ranging discussions, and summarizes major areas of both consensus and disagreement, as well research topics that are likely to be high priorities in the years to come.

Neurochemical characterization of the release and uptake of dopamine in ventral tegmental area and serotonin in substantia nigra of the mouse.

In the present report, fast-scan cyclic voltammetry was used to identify the monoamines that were released by electrical stimulation in mouse brain slices containing ventral tegmental area (VTA), substantia nigra (SN) -pars compacta (SNc) and -pars reticulata (SNr). We showed that voltammograms obtained in mouse VTA were consistent with detection of a catecholamine, while those in both subregions of the SN were consistent with detection of an indolamine, based on the reduction peak potentials. We used pharmacological blockade and genetic deletion of monoamine transporters to further confirm the identity of released monoamines in mouse midbrain and to assess the control of monoamines by their transporters in each brain region. Inhibition of dopamine and norepinephrine transporters by nomifensine (1 and 10 microm) decreased uptake rates in the VTA, but did not change uptake rates in either subregion of the SN. Serotonin transporter inhibition by fluoxetine (10 microm) decreased uptake rates in the SNc and SNr, but was without effect in the VTA. Selective inhibition of the norepinephrine transporter by desipramine (10 microm) had no effect in any brain region. Using dopamine transporter- and serotonin transporter-knockout mice, we found decreased uptake rates in VTA and SN subregions, respectively. Peak signals recorded in each midbrain region were pulse number dependent and exhibited limited frequency dependence. Thus, dopamine is predominately detected by voltammetry in mouse VTA, while serotonin is predominately detected in mouse SNc and SNr. Furthermore, active uptake occurs in these areas and can be altered only by specific uptake inhibitors, suggesting a lack of heterologous uptake. In addition, somatodendritic dopamine release in VTA was not mediated by monoamine transporters. This work offers an initial characterization of voltammetric signals in the midbrain of the mouse and provides insight into the regulation of monoamine neurotransmission in these areas.

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.

Role of serotonin in cocaine effects in mice with reduced dopamine transporter function.

The mesolimbic dopaminergic system, especially the nucleus accumbens, has received attention for its involvement in the reinforcing and addictive properties of cocaine and other drugs of abuse. It is generally accepted that the ability of cocaine to inhibit the dopamine transporter (DAT) is directly related to its reinforcing actions. However, mice with a genetic deletion of the DAT (DAT-KO mice) still experience the rewarding effects of cocaine. These behavioral findings suggest that there is an alternate site for cocaine reinforcement. We demonstrate here that modulation of the serotonergic system in the ventral tegmental area, where the mesolimbic dopamine system originates, is a target of cocaine action. The ultimate effect of this serotonin mechanism in animal models with sustained elevations of dopamine may be a feed-forward enhancement of dopamine levels in the nucleus accumbens.

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.