Novel Allosteric Modulator Southern Research Institute-32743 Reverses HIV-1 Transactivator of Transcription-Induced Increase in Dopamine Release in the Caudate Putamen of Inducible Transactivator of Transcription Transgenic Mice.

Development of neurocognitive disorder in human immunodeficiency virus (HIV)-infected patients has been linked to dysregulation of dopamine by the HIV-1 transactivator of transcription (Tat) protein, a negative allosteric modulator of dopamine transporter (DAT). Using fast scan cyclic voltammetry, the present study determined the effects of in vivo Tat expression on dopamine release in the caudate putamen of inducible Tat transgenic (iTat-tg) mice and the impact of a novel DAT allosteric modulator, Southern Research Institute (SRI)-32743, on the Tat effect. We found that 7- or 14-day doxycycline (Dox)-induced Tat expression in iTat-tg mice resulted in a 2-fold increase in phasic but not tonic stimulated baseline dopamine release relative to saline control mice. To determine whether the Tat-induced increase in dopamine release is mediated by DAT regulation, we examined the effect of an in vitro applied DAT inhibitor, nomifensine, on the dopamine release. Nomifensine (1 nM-10 µM) concentration-dependently enhanced phasic stimulated dopamine release in both saline- and Dox-treated iTat-tg mice, while the magnitude of the nomifensine-mediated dopamine release was unchanged between saline and Dox treatment groups. A single systemic administration of SRI-32743 prior to animal sacrifice reversed the increased dopamine release in the baseline of phasic dopamine release and nomifensine-augmented dopamine levels in Dox-treated iTat-tg mice, while SRI-32743 alone did not alter baseline of dopamine release. These findings suggest that Tat expression induced an increase in extracellular dopamine levels by not only inhibiting DAT-mediated dopamine transport but also stimulating synaptic dopamine release. Thus, DAT allosteric modulators may serve as a potential therapeutic intervention for HIV infection-dysregulated dopamine system observed in HIV-1 positive individuals. SIGNIFICANCE STATEMENT: HIV infection-induced dysregulation of the dopaminergic system has been implicated in the development of neurocognitive impairments observed in HIV positive patients. Understanding the mechanisms underlying HIV-1 Tat protein-induced alteration of extracellular dopamine levels will provide insights into the development of molecules that can attenuate Tat interaction with targets in the dopaminergic system. Here, we determined whether Tat alters dopamine release and how the novel DAT allosteric modulator, SRI-32743, impacts dopamine neurotransmission to attenuate Tat-induced effects on extracellular dopamine dynamics.

Structure-Based Discovery of a Novel Allosteric Inhibitor against Human Dopamine Transporter.

Human dopamine transporter (hDAT) regulates the reuptake of extracellular dopamine (DA) and is an essential therapeutic target for central nervous system (CNS) diseases. The allosteric modulation of hDAT has been identified for decades. However, the molecular mechanism underlying the transportation is still elusive, which hinders the rational design of allosteric modulators against hDAT. Here, a systematic structure-based method was performed to explore allosteric sites on hDAT in inward-open (IO) conformation and to screen compounds with allosteric affinity. First, the model of the hDAT structure was constructed based on the recently reported Cryo-EM structure of the human serotonin transporter (hSERT) and Gaussian-accelerated molecular dynamics (GaMD) simulation was further utilized for the identification of intermediate energetic stable states of the transporter. Then, with the potential druggable allosteric site on hDAT in IO conformation, virtual screening of seven enamine chemical libraries (∼440,000 compounds) was processed, resulting in 10 compounds being purchased for in vitro assay and with Z1078601926 discovered to allosterically inhibit hDAT (IC50 = 0.527 [0.284; 0.988] µM) when nomifensine was introduced as an orthosteric ligand. Finally, the synergistic effect underlying the allosteric inhibition of hDAT by Z1078601926 and nomifensine was explored using additional GaMD simulation and postbinding free energy analysis. The hit compound discovered in this work not only provides a good starting point for lead optimization but also demonstrates the usability of the method for the structure-based discovery of novel allosteric modulators of other therapeutic targets.

Neurotoxic effects of exposure to glyphosate in rat striatum: Effects and mechanisms of action on dopaminergic neurotransmission.

The main objective of this study was to evaluate the effects and possible mechanisms of action of glyphosate and a glyphosate-based herbicide (GBH) on dopaminergic neurotransmission in the rat striatum. Acute exposure to glyphosate or GBH, administered by systemic (75 or 150 mg/kg, i.p.) or intrastriatal (1, 5, or 10 mM for 1 h) routes, produced significant concentration-dependent increases in dopamine release measured in vivo by cerebral microdialysis coupled to HPLC with electrochemical detection. Systemic administration of glyphosate also significantly impaired motor control and decreased striatal acetylcholinesterase activity and antioxidant capacity. At least two mechanisms can be proposed to explain the glyphosate-induced increases in extracellular dopamine levels: increased exocytotic dopamine release from synaptic vesicles or inhibition of dopamine transporter (DAT). Thus, we investigated the effects of intrastriatal administration of glyphosate (5 mM) in animals pretreated with tetrodotoxin (TTX) or reserpine. It was observed that TTX (10 or 20 µM) had no significant effect on glyphosate-induced dopamine release, while reserpine (10 mg/kg i.p) partially but significantly reduced the dopamine release. When glyphosate was coinfused with nomifensine (50 µM), the increase in dopamine levels was significantly higher than that observed with glyphosate or nomifensine alone. So, two possible hypotheses could explain this additive effect: both glyphosate and nomifensine act through different mechanisms at the dopaminergic terminals to increase dopamine levels; or both nomifensine and glyphosate act on DAT, with glyphosate simultaneously inhibiting reuptake and stimulating dopamine release by reversing the DAT function. Future research is needed to determine the effects of this pesticide at environmentally relevant doses.

In Vivo Monitoring of Dopamine by Microdialysis with 1 min Temporal Resolution Using Online Capillary Liquid Chromatography with Electrochemical Detection.

Microdialysis is often applied to understanding brain function. Because neurotransmission involves rapid events, increasing the temporal resolution of in vivo measurements is desirable. Here, we demonstrate microdialysis with online capillary liquid chromatography for the analysis of 1 min rat brain dialysate samples at 1 min intervals. Mobile phase optimization involved adjusting the pH, buffer composition, and surfactant concentration to eliminate interferences with the dopamine peak. By analyzing electrically evoked dopamine transients carefully synchronized with the switching of the online LC sample valve, we demonstrate that our system has both 1 min sampling capabilities and bona fide 1 min temporal resolution. Evoked DA transients were confined to single, 1 min brain dialysate samples. After uptake inhibition with nomifensine (20 mg/kg i.p.), responses to electrical stimuli of 1 s duration were detected.

Impaired neurotransmission caused by overexpression of α-synuclein in nigral dopamine neurons.

We used in vivo amperometry to monitor changes in synaptic dopamine (DA) release in the striatum induced by overexpression of human wild-type α-synuclein in nigral DA neurons, induced by injection of an adeno-associated virus type 6 (AAV6)-α-synuclein vector unilaterally into the substantia nigra in adult rats. Impairments in DA release evolved in parallel with the development of degenerative changes in the nigrostriatal axons and terminals. The earliest change, seen 10 d after vector injection, was a marked, ≈50%, reduction in DA reuptake, consistent with an early dysfunction of the DA transporter that developed before any overt signs of axonal damage. At 3 wk, when the first signs of axonal damage were observed, the amount of DA released after a KCl pulse was reduced by 70-80%, and peak DA concentration was delayed, indicating an impaired release mechanism. At later time points, 8-16 wk, overall striatal innervation density was reduced by 60-80% and accompanied by abundant signs of axonal damage in the form of α-synuclein aggregates, axonal swellings, and dystrophic axonal profiles. At this stage DA release and reuptake were profoundly reduced, by 80-90%. The early changes in synaptic DA release induced by overexpression of human α-synuclein support the idea that early predegenerative changes in the handling of DA may initiate, and drive, a progressive degenerative process that hits the axons and terminals first. Synaptic dysfunction and axonopathy would thus be the hallmark of presymptomatic and early-stage Parkinson disease, followed by neuronal degeneration and cell loss, characteristic of more advanced stages of the disease.

Region- and domain-dependent action of nomifensine.

The dopamine (DA) terminal fields in the rat dorsal striatum (DS) and nucleus accumbens core (NAcc) are organized as patchworks of domains that exhibit distinct kinetics of DA release and clearance. The present study used fast-scan cyclic voltammetry recordings of electrically evoked DA overflow to test the hypothesis that nomifensine might exhibit domain-dependent actions within the NAcc, as we previously found to be the case within the DS. Within the NAcc, nomifensine preferentially enhanced evoked DA overflow in the slow domains compared with the fast domains. To seek a kinetic explanation for nomifensine's selective actions, we quantified the apparent KM of DA clearance by numerically evaluating the derivative of the descending phase of the DA signal after the end of the stimulus. For comparison, we likewise quantified the apparent KM in the domains of the DS. As expected, because it is a competitive inhibitor, nomifensine significantly increased the apparent KM in both the fast and slow domains of both the NAcc and DS. However, our analysis also led to the novel finding that nomifensine preferentially increases the apparent KM in the NAcc compared with the DS; the apparent KM increased by ~500% in the NAcc and by ~200% in the DS.

Diagnostic pitfalls of drug-induced immune hemolytic anemia.

Immune hemolytic anemia (IHA) is a rare complication of drug administration. However, its true incidence remains obscure, as there are a number of factors that may lead to misdiagnosis. The clinical and serologic pictures are variable, and there is a great deal of unawareness that certain drugs can cause IHA. Furthermore, serologic results can be easily misinterpreted, resulting in a wrong diagnosis.

Parallel on-chip analysis of single vesicle neurotransmitter release.

Real-time investigations of neurotransmitter release provide a direct insight on the mechanisms involved in synaptic communication. Carbon fiber microelectrodes are state-of-the-art tools for electrochemical measurements of single vesicle neurotransmitter release. Yet, they lack high-throughput capabilities that are required for collecting robust statistically significant data across multiple samples. Here, we present a chip-based recording system enabling parallel in vitro measurements of individual neurotransmitter release events from cells, cultured directly on planar multielectrode arrays. The applicability of this cell-based platform to pharmacological screening is demonstrated by resolving minute concentration-dependent effects of the dopamine reuptake inhibitor nomifensine on recorded single-vesicle release events from PC12 cells. The experimental results, showing an increased half-time of the recorded events, are complemented by an analytical model for the verification of drug action.

Restoration of dopamine release deficits during object recognition memory acquisition attenuates cognitive impairment in a triple transgenic mice model of Alzheimer's disease.

Previous findings indicate that the acquisition and consolidation of recognition memory involves dopaminergic activity. Although dopamine deregulation has been observed in Alzheimer's disease (AD) patients, the dysfunction of this neurotransmitter has not been investigated in animal models of AD. The aim of this study was to assess, by in vivo microdialysis, cortical and hippocampal dopamine, norepinephrine, and glutamate release during the acquisition of object recognition memory (ORM) in 5- and 10-mo-old triple-transgenic Alzheimer's disease mice (3xTg-AD) and to relate the extracellular changes to 24-h memory performance. Five- and 10-mo-old wild-type mice and 5-mo-old 3xTg-AD showed significant cortical but not hippocampal dopamine increase during object exploration. On a 24-h ORM test, these three groups displayed significant ORM. In contrast, 10-mo-old 3xTg-AD mice showed impaired dopamine release in the insular cortex during ORM acquisition, as well as significant impairment in ORM. In addition, cortical administration of a dopamine reuptake blocker produced an increase of dopamine levels in the 10-mo-old 3xTg-AD mice and attenuated the memory impairment. These data suggest that activation of the dopaminergic system in the insular cortex is involved in object recognition memory, and that dysfunction of this system contributes to the age-related decline in cognitive functioning of the 3xTg-AD mice.

Modulation of nigral dopamine signaling mitigates parkinsonian signs of aging: evidence from intervention with calorie restriction or inhibition of dopamine uptake.

Identifying neurobiological mechanisms of aging-related parkinsonism, and lifestyle interventions that mitigate them, remain critical knowledge gaps. No aging study, from rodent to human, has reported loss of any dopamine (DA) signaling marker near the magnitude associated with onset of parkinsonian signs in Parkinson's disease (PD). However, in substantia nigra (SN), similar loss of DA signaling markers in PD or aging coincide with parkinsonian signs. Alleviation of these parkinsonian signs may be possible by interventions such as calorie restriction (CR), which augment DA signaling markers like tyrosine hydroxylase (TH) expression in the SN, but not striatum. Here, we interrogated respective contributions of nigral and striatal DA mechanisms to aging-related parkinsonian signs in aging (18 months old) rats in two studies: by the imposition of CR for 6 months, and inhibition of DA uptake within the SN or striatum by cannula-directed infusion of nomifensine. Parkinsonian signs were mitigated within 12 weeks after CR and maintained until 24 months old, commensurate with increased D1 receptor expression in the SN alone, and increased GDNF family receptor, GFR-α1, in the striatum, suggesting increased GDNF signaling. Nomifensine infusion into the SN or striatum selectively increased extracellular DA. However, only nigral infusion increased locomotor activity. These results indicate mechanisms that increase components of DA signaling in the SN alone mitigate parkinsonian signs in aging, and are modifiable by interventions, like CR, to offset parkinsonian signs, even at advanced age. Moreover, these results give evidence that changes in nigral DA signaling may modulate some parameters of locomotor activity autonomously from striatal DA signaling.

Domain-dependent effects of DAT inhibition in the rat dorsal striatum.

The rat dorsal striatum exhibits domain-dependent kinetics of dopamine release and clearance. The present report describes the domain-dependent actions of nomifensine (20 mg/kg i.p.), a competitive dopamine uptake inhibitor, on evoked dopamine responses recorded by voltammetry during electrical stimulation of the medial forebrain bundle. In slow domains, nomifensine increases the initial rate of evoked overflow, increases response overshoot, does not affect the slope of the linear segment of the dopamine clearance profile, and slows the non-linear segment of the clearance profile. In fast domains, nomifensine does not affect the initial rate of overflow, increases the end-of-stimulus overshoot, and decreases the slope of the linear segment of the dopamine clearance profile. Collectively, these findings do not concur with existing models of evoked dopamine release that describe the effect of nomifensine as an increase in the effective KM of dopamine uptake. These findings suggest that dopamine clearance after evoked release is affected by both dopamine uptake and a restricted extracellular diffusion process.

Dopamine in the hippocampus is cleared by the norepinephrine transporter.

Abnormal dopaminergic neurotransmission in the hippocampus may be involved in certain aspects of cognitive dysfunction. In the hippocampus, there is little, if any, expression of dopamine transporters (DAT), indicating that the mechanism for dopamine clearance differs from that in the striatum. Here, by means of in-vivo microdialysis in freely moving rats, we tested the hypothesis that the norepinephrine transporter (NET) is involved in dopamine clearance in the hippocampus. We found that systemic administration of the selective NET inhibitor reboxetine (3 mg/kg) and the psychostimulants amphetamine (0.5 mg/kg) and cocaine (10 mg/kg) increased hippocampal dopamine efflux. Local administration of reboxetine (300 µM) produced a large increase in hippocampal dopamine levels that could not be further enhanced by the addition of the NET/DAT inhibitor nomifensine (100 µM). Administration of the specific DAT inhibitor GBR12909 at a concentration (1 mM) that robustly increased dopamine in the nucleus accumbens had a comparably smaller effect in the hippocampus. In line with a minor role of DAT in the hippocampus, we detected very little DAT in this area using ligand binding with radiolabelled RTI-55. Moreover, in contrast to raclopride (100 µM), a dopamine D2-autoreceptor antagonist, local administration of the α2-adrenoceptor antagonist idazoxan (100 µM) increased hippocampal dopamine. Taken together, our data demonstrate an interaction between dopamine and norepinephrine systems in the hippocampus. It is proposed that this interaction originates from a shared uptake mechanism at the NET level.

Nomifensine alters sex differences in striatal dopaminergic function.

A series of three experiments are presented in which the acute effects of the catecholamine reuptake inhibitor, nomifensine, upon striatal dopaminergic function are compared in female and male mice. In Experiment 1, treatment with nomifensine (5 mg kg⁻¹), at 30 min prior to injection of methamphetamine (40 mg kg⁻¹) significantly decreased the amount of striatal dopamine depletion in male, but not female, mice, thereby abolishing the sex difference in methamphetamine-induced neurotoxicity (males > females). In Experiment 2, the methamphetamine-evoked sex differences in dopamine and DOPAC output from superfused striatal tissue (males > females) were abolished in mice treated with nomifensine at 30 min prior to tissue removal. In Experiment 3, the potassium chloride-evoked sex differences in dopamine and DOPAC output from superfused striatal tissue (females > males) were reversed in mice treated with nomifensine at 30 min prior to tissue removal. Taken together these results demonstrate the critical role played by catecholamine transporters in sex differences of dopaminergic function and suggest that this may involve the dopamine transporter, due to its high concentrations within the striatum. Such findings highlight the need for gender-specific considerations in use of treatments that target reuptake transporters function.

Electrokinetic chromatographic estimation of the enantioselective binding of nomifensine to human serum albumin and total plasma proteins.

This report is the first evidence of enantioselective binding of nomifensine to human serum albumin (HSA) and plasma proteins. The overall process with HSA included: (i) consistent experimental design along two independent sessions; (ii) incubation of nomifensine-HSA designed mixtures; (iii) ultrafiltration for separating the unbound enantiomers fraction; (iv) electrokinetic chromatography (EKC) using heptakis-2,3,6-tri-O-methyl-ß-cyclodextrin as chiral selector to provide experimental data for enantiomers (first, E1, and second, E2, eluted ones); and (v) a recent direct equation allowing univariate tests and robust statistics to provide consistent parameters and uncertainty. A significant enantioselectivity to HSA (2.7 ± 0.1) was encountered, related to a 1:1 stoichiometry and log affinity constants of 3.24 ± 0.10 and 3.67 ± 0.08 for E1 and E2, respectively. The protein binding (PB) estimated at physiological concentration levels was 40 ± 5 and 63 ± 4% for E1 and E2, respectively. The use of synthetic human sera allowed in vitro estimation of the total plasma PB for the racemate (61 ± 5%; coincident with in vivo values), and its enantiomers (58 ± 7 and 64 ± 4% for E1 and E2, respectively). Comparison allowed the relative importance of HSA respect to other plasma proteins for binding nomifensine to be established.

Spectrofluorimetric determination of nomifensine in human plasma and urine by derivatization with fluorescamine.

A sensitive, simple and rapid spectrofluorimetric method was developed for the determination of nomifensine in human plasma and urine. The present method was based on the derivatization by fluorescamine in phosphate buffer at pH 4.0 to produce a highly fluorescent product which was measured at 488 nm (excitation at 339 nm). The method was validated according to the ICH guidelines with respect to linearity, limit of detection, limit of quantification, accuracy, precision, recovery and robustness. The assay was linear over the concentration ranges 100-2,000 and 50-2,000 ng/mL for plasma and urine, respectively. The limits of detection were calculated to be 13.9 and 7.5 ng/mL for plasma and urine, respectively. The method was successfully applied to the analysis of the drug in human plasma and urine.

mPFC catecholamines modulate attentional capture by appetitive distracters and attention to time in a peak-interval procedure in rats.

The behavioral and neural mechanisms by which distracters delay interval timing behavior are currently unclear. Distracters delay timing in a considerable dynamic range: Some distracters have no effect on timing ("run"), whereas others seem to "stop" timing; some distracters restart ("reset") the entire timing mechanisms at their offset, whereas others seem to capture attentional resources long after their termination ("over-reset"). While the run-reset range of delays is accounted for by the Time-Sharing Hypothesis (Buhusi, 2003, 2012), the behavioral and neural mechanisms of "over-resetting" are currently uncertain. We investigated the role of novelty (novel/familiar) and significance (consequential/inconsequential) in the time-delaying effect of distracters and the role of medial prefrontal cortex (mPFC) catecholamines by local infusion of norepinephrine-dopamine reuptake inhibitor (NDRI) nomifensine in a peak-interval (PI) procedure in rats. Results indicate differences in time delay between groups, suggesting a role for both novelty and significance: inconsequential, familiar distracters "stopped" timing, novel distracters "reset" timing, whereas appetitively conditioned distracters "over-reset" timing. mPFC infusion of nomifensine modulated attentional capture by appetitive distracters in a "U"-shaped fashion, reduced the delay after novel distracters, but had no effects after inconsequential, familiar distracters. These results were not due to nomifensine affecting either timing accuracy, precision, or peak response rate. Results may help elucidate the behavioral and physiological mechanisms underlying interval timing and attention to time and may contribute to developing new treatment strategies for disorders of attention. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Characterization of D3 Autoreceptor Function in Whole Zebrafish Brain with Fast-Scan Cyclic Voltammetry.

Zebrafish (Danio rerio) are ideal model organisms for investigating nervous system function, both in health and disease. Nevertheless, functional characteristics of dopamine (DA) release and uptake regulation are still not well-understood in zebrafish. In this study, we assessed D3 autoreceptor function in the telencephalon of whole zebrafish brains ex vivo by measuring the electrically stimulated DA release ([DA]max) and uptake at carbon fiber microelectrodes with fast-scan cyclic voltammetry. Treatment with pramipexole and 7-OH-DPAT, selective D3 autoreceptor agonists, sharply decreased [DA]max. Conversely, SB277011A, a selective D3 antagonist, nearly doubled [DA]max and decreased k, the first-order rate constant for the DA uptake, to about 20% of its original value. Treatment with desipramine, a selective norepinephrine transporter blocker, failed to increase current, suggesting that our electrochemical signal arises solely from the release of DA. Furthermore, blockage of DA uptake with nomifensine-reversed 7-OH-DPAT induced decreases in [DA]max. Collectively, our data show that, as in mammals, D3 autoreceptors regulate DA release, likely by inhibiting uptake. The results of this study are useful in the further development of zebrafish as a model organism for DA-related neurological disorders such as Parkinson's disease, schizophrenia, and drug addiction.

Sigma receptor ligands haloperidol and ifenprodil attenuate hypoxia induced dopamine release in rat striatum.

OBJECTIVE: We aimed to investigate the hypothesis that sigma receptor ligands, haloperidol and ifenprodil, attenuate hypoxia-induced striatal dopamine release in vitro and determine the possible mechanisms. METHODS: Extracellular concentrations of dopamine were measured using acute brain slices method under hypoxic, aglycemic and ischemic conditions. Sigma receptor ligands haloperidol and ifenprodil attenuate striatal dopamine release induced by hypoxia in contrast to aglycemia and ischemia. To determine the possible contribution of glutamatergic system on this effect, we compared the effect of NMDA receptor antagonist MK-801 and haloperidol in hypoxia induced by Na-K-ATPaz enzyme inhibitor ouabain. Also, we compared the effect of dopamine uptake blocker nomifensine and haloperidol to determine the role of dopamine transporter on this effect. RESULTS: Haloperidol and nomifensine almost completely abolish ouabain-induced dopamine release unlike MK-801. Different effects of sigma ligands and glutamate receptor antagonists on the hypoxia and ouabain induced dopamine release show that glutamate receptor blockade is partial involved in inhibitory effect of sigma ligand on dopamine release under hypoxic conditions. Similar effect of dopamine uptake blocker nomifensine and sigma receptor ligand haloperidol on ouabain induced dopamine release supports the possibility that inhibition of reverse dopamine transport by sigma ligands might be involved in their protective effect. CONCLUSIONS: Data in this study suggest that sigma ligands may be a new therapeutic intervention for the management of hypoxic conditions.

SKF-83566, a D1-dopamine receptor antagonist, inhibits the dopamine transporter.

Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1)-receptor activation in axonal DA release regulation in dorsal striatum using a D(1)-receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of ∼ 65% in 5 µM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 µM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3)H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 µM. Binding studies with [(3)H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 µM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT.

Inhibition of dopamine uptake by D2 antagonists: an in vivo study.

D(2)-like antagonists potentiate dopamine release. They also inhibit dopamine uptake by a mechanism yet to be clarified. Here, we monitored dopamine uptake in the striatum of anesthetized mice. The dopamine overflow was evoked by brief electrical stimulation of the medial forebrain bundle (four pulses at 100 Hz) and was monitored with carbon fiber electrodes combined with continuous amperometry. The decay phase of evoked overflows reflects dopamine half-life, which entirely depends on uptake. The D(2)-like antagonists haloperidol and eticlopride enhanced the half-life by 45% and 48%, respectively, a moderate effect as compared to the uptake blocker nomifensine (528%). Both D(2)-like antagonists did not affect dopamine uptake in mice lacking D(2) receptors. Inhibition of tonic dopamine release by gamma-butyrolactone did not mimic the enhancing effect of D(2) antagonists on dopamine half-life. However, prolonged stimulation boosted dopamine uptake and this effect was not observed after haloperidol treatment or in mice lacking D(2) receptors. Therefore, dopamine uptake is accelerated in conditions of excessive D(2) stimulation but not finely tuned in resting conditions. Inhibition of dopamine uptake by D(2) antagonists synergizes with the potentiation of dopamine release to strongly alter the phasic dopamine signaling.