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.

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.

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.

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.

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).

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.

A high fat western diet attenuates phasic dopamine release.

Natural rewards, such as food and social interaction, as well as drugs of abuse elicit increased mesolimbic dopamine release in the nucleus accumbens (NAc). Drugs of abuse, however, increase NAc dopamine release to a greater extent and are known to induce lasting changes on the functioning of the mesolimbic dopamine pathway. Less is known about the long-term effects of diet composition on this reward pathway. In the present study, two diets were compared: a higher-fat diet (Western Diet: WD) and a control diet (standard lab chow) on their effect on the mesolimbic dopamine system. Twenty male C57BL/6 J mice were placed on one of these diets at 7 weeks old. After twelve weeks on the diet, in vivo fixed potential amperometry was used to measure real-time stimulation-evoked dopamine release in the NAc of anesthetized mice before and after an i.p. injection of the dopamine transporter (DAT) inhibitor nomifensine. Results indicated that diet altered mesolimbic dopamine functioning. Mice that consumed the WD demonstrated a hypodopaminergic profile, specifically reduced baseline dopamine release and an attenuated dopaminergic response to DAT inhibition compared to the control diet group. Thus, diet may play a role in mediating dopamine-related behavior, disorders associated with dopamine dysfunction, and pharmacological treatments aimed at altering dopamine transmission.

Protection of dopamine neurons by CDNF and neurturin variant N4 against MPP+ in dissociated cultures from rat mesencephalon.

Parkinson's disease is associated with the loss of dopamine (DA) neurons in ventral mesencephalon. We have previously reported that no single neurotrophic factor we tested protected DA neurons from the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP+) in dissociated cultures isolated from the P0 rat substantia nigra, but that a combination of five neurotrophic factors was protective. We now report that cerebral DA neurotrophic factor (CDNF) and a variant of neurturin (NRTN), N4, were also not protective when provided alone but were protective when added together. In cultures isolated from the substantia nigra, MPP+ (10 µM) decreased tyrosine hydroxylase-positive cells to 41.7 ± 5.4% of vehicle control. Although treatment of cultures with 100 ng/ml of either CDNF or N4 individually before and after toxin exposure did not significantly increase survival in MPP+-treated cultures, when the two trophic factors were added together at 100 ng/ml each, survival of cells was increased 28.2 ± 6.1% above the effect of MPP+ alone. In cultures isolated from the ventral tegmental area, another DA rich area, a higher dose of MPP+ (1 mM) was required to produce an EC50 in TH-positive cells but, as in the substantia nigra, only the combination of CDNF and N4 (100 ng/ml each) was successful at increasing the survival of these cells compared to MPP+ alone (by 22.5 ± 3.5%). These data support previous findings that CDNF and N4 may be of therapeutic value for treatment of PD, but suggest that they may need to be administered together.

Triple reuptake inhibition of serotonin, norepinephrine, and dopamine increases the tonic activation of α2-adrenoceptors in the rat hippocampus and dopamine levels in the nucleus accumbens.

Clinical studies have shown the therapeutic efficacy of an increase in dopamine (DA) transmission in treatment of major depressive disorder (MDD). In the present study, we investigated whether blockade of DA transporters in addition to serotonin (5-HT) and norepinephrine (NE) produced additional adaptations of monoaminergic systems. In vivo electrophysiological recordings were carried out in male anesthetized rats. Vehicle, the 5-HT reuptake inhibitor escitalopram, the NE/DA reuptake blocker nomifensine and their combination (triple reuptake inhibition; TRI) were delivered for 2 or 14 days. Firing activity of NE, 5-HT and DA neurons was assessed. Tonic activation of 5-HT1A receptors and α1- and α2-adrenoceptors was determined in the hippocampus and extracellular DA levels in the nucleus accumbens (NAc). Unlike escitalopram, nomifensine and TRI administration increased the tonic activation of α2-adrenoceptors in the hippocampus despite decreasing NE neuronal firing activity after 2 and 14 days of administration. The firing activity of 5-HT neurons was increased after prolonged nomifensine and TRI regimens, while addition of nomifensine to escitalopram prevented the early 2-day suppression of firing by 5-HT reuptake inhibition. The tonic activation of 5-HT1A receptors was enhanced only with escitalopram. Whereas escitalopram and nomifensine decreased firing activity of DA neurons after a 2-day administration, their combination normalized it to baseline level after 14 days; this was accompanied by a robust increase in extracellular DA levels in the NAc. In summary, these results indicate that TRI increases NE and DA but not 5-HT transmission, suggesting a differential efficacy profile in MDD patients.

Systemic oxytocin administration alters mesolimbic dopamine release in mice.

Growing research indicates oxytocin may be involved in relieving anxiety and attenuating the rewarding effects of psychostimulants. This study investigated the effects of subchronic oxytocin treatments on mesolimbic dopamine transmission in areas associated with anxiety and addiction, the amygdala and the nucleus accumbens (NAc), respectively. Using in vivo fixed potential amperometry, stimulation-evoked dopamine release was recorded in anesthetized mice pretreated with subchronic oxytocin (four i.p. injections of 1 mg/kg oxytocin or saline with 48 h between injections). During dopamine recordings, mice received an i.p. drug challenge of either oxytocin (1 mg/kg), the dopamine reuptake blocker nomifensine (10 mg/kg), or saline. Overall, subchronic oxytocin pretreatment did alter properties of dopamine release in these limbic structures. In the amygdala, dopamine release was decreased following the oxytocin challenge but only in oxytocin pretreated mice. In the NAc, baseline dopamine release was attenuated in oxytocin pretreated mice relative to saline pretreated mice. Furthermore, oxytocin pretreated mice displayed a reduced dopaminergic response to the drug challenge of nomifensine relative to control mice. Together these results suggest that oxytocin may be useful at treating aspects of anxiety and drug abuse. Elucidating the neural effects of oxytocin is critical given the multitude of potential therapeutic uses for this drug.

Electrophysiological Characterization of Novel Effects of the Uptake-2 Blocker Decynium-22 (D-22) on Dopaminergic Neurons in the Substantia Nigra Pars Compacta.

Extracellular levels of dopamine (DA) and other monoamines in the brain depend not only on the classic transporters encoded by SLC6A gene family such as DAT, NET and SERT, but also a more recently identified group of low-affinity/high-capacity 'Uptake-2' transporters, mainly OCT3 and PMAT. The most frequently used pharmacological tool in functional studies of Uptake-2 is decynium-22 (D-22) known to block these transporters. However, the effectiveness of this drug in enhancing extracellular DA remains uncertain. Our aim was to test the hypothesis that D-22 increases extracellular levels of DA released from the somatodendritic region of dopaminergic neurons in the substantia nigra pars compacta (SNc) by reducing the OCT3/PMAT-dependent component of DA uptake. Extracellular DA was assessed indirectly, by evoking D2-IPSCs in SNc neurons following stimulated release of this neurotransmitter in midbrain slices obtained from mice. Recordings were conducted after partial inhibition of DAT with nomifensine, and after application of L-DOPA which increased the releasable DA pool. Contrary to our expectations, D-22 reduced, rather than increased, the amplitude of D2-IPSCs. Other effects included inhibition of GABAB-IPSCs and Ih current, and a reduction in firing frequency of nigral neurons. These results show that in addition to the previously known non-specific inhibitory action on α1 adrenoceptors, D-22 exerts additional off-target effects by inhibiting dopaminergic and GABAergic synaptic transmission in the SNc and the spontaneous (pacemaker) activity of nigral neurons. It remains to be established if these novel effects contribute to a reduction in spontaneous locomotor activity reported in previous studies after systemic drug administration.

Dopamine release in mushroom bodies of the honey bee (Apis mellifera L.) in response to aversive stimulation.

In Drosophila melanogaster, aversive (electric shock) stimuli have been shown to activate subpopulations of dopaminergic neurons with terminals in the mushroom bodies (MBs) of the brain. While there is compelling evidence that dopamine (DA)-induced synaptic plasticity underpins the formation of aversive memories in insects, the mechanisms involved have yet to be fully resolved. Here we take advantage of the accessibility of MBs in the brain of the honey bee to examine, using fast scan cyclic voltammetry, the kinetics of DA release and reuptake in vivo in response to electric shock, and to investigate factors that modulate the release of this amine. DA increased transiently in the MBs in response to electric shock stimuli. The magnitude of release varied depending on stimulus duration and intensity, and a strong correlation was identified between DA release and the intensity of behavioural responses to shock. With repeated stimulation, peak DA levels increased. However, the amount of DA released on the first stimulation pulse typically exceeded that evoked by subsequent pulses. No signal was detected in response to odour alone. Interestingly, however, if odour presentation was paired with electric shock, DA release was enhanced. These results set the stage for analysing the mechanisms that modulate DA release in the MBs of the bee.

Smaller effect of propofol than sevoflurane anesthesia on dopamine turnover induced by methamphetamine and nomifensine in the rat striatum: an in vivo microdialysis study.

Volatile anesthetics accelerate dopamine turnover in the brain, especially when used in conjunction with psychotropic agents such as methamphetamine and nomifensine. The effect of intravenous propofol anesthesia on the extracellular dopamine concentrations is unclear. The aim of this study was to compare the effect of two anesthetics on the extracellular concentrations of dopamine and metabolites using an in vivo microdialysis model. Male Sprague Dawley rats were implanted with a microdialysis probe into the right striatum. The probe was perfused with modified Ringer's solution, and the dialysate was directly injected into a high-performance liquid chromatography system every 20 min. The rats were intraperitoneally administered saline, methamphetamine at 2 mg/kg, or nomifensine at 10 mg/kg. After treatment, the rats were anesthetized with intravenous propofol (20 mg/kg followed by 25 or 50 mg/kg/h) or inhalational sevoflurane (2.5%) for 1 h. Propofol showed no effect on the extracellular concentration of dopamine during anesthesia; however, propofol decreased the dopamine concentration after anesthesia in the high-dose group. Sevoflurane anesthesia increased the concentration of metabolites. Systemic administration of methamphetamine and nomifensine increased the extracellular concentration of dopamine. Sevoflurane anesthesia significantly enhanced the increase in the dopamine concentration induced by both methamphetamine and nomifensine, whereas propofol anesthesia showed no effect on the methamphetamine- and nomifensine-induced dopamine increase during anesthesia. The enhancing effect of psychotropic agent-induced acceleration of dopamine turnover was smaller for propofol anesthesia than for sevoflurane anesthesia.

Monitoring Dopamine Responses to Potassium Ion and Nomifensine by in Vivo Microdialysis with Online Liquid Chromatography at One-Minute Resolution.

Recently, our laboratory has demonstrated the technical feasibility of monitoring dopamine at 1 min temporal resolution with microdialysis and online liquid chromatography. Here, we monitor dopamine in the rat striatum during local delivery of high potassium/low sodium or nomifensine in awake-behaving rats. Microdialysis probes were implanted and perfused continuously with or without dexamethasone in the perfusion fluid for 4 days. Dexamethasone is an anti-inflammatory agent that exhibits several positive effects on the apparent health of the brain tissue surrounding microdialysis probes. Dopamine was monitored 1 or 4 days after implantation under basal conditions, during 10 min applications of 60 mM or 100 mM K+, and during 15 min applications of 10 µM nomifensine. High K+ and nomifensine were delivered locally by adding them to the microdialysis perfusion fluid using a computer-controlled, low-dead-volume six-port valve. Each day/K+/dexamethasone combination elicited specific dopamine responses. Dexamethasone treatment increased dopamine levels in basal dialysates (i.e., in the absence of K+ or nomifensine). Applications of 60 mM K+ evoked distinct responses on days one and four after probe implantation, depending upon the presence or absence of dexamethasone, consistent with dexamethasone's ability to mitigate the traumatic effect of probe implantation. Applications of 100 mM K+ evoked dramatic oscillations in dopamine levels that correlated with changes in the field potential at a metal electrode implanted adjacent to the microdialysis probe. This combination of results indicates the role of spreading depolarization in response to 100 mM K+. With 1 min temporal resolution, we find that it is possible to characterize the pharmacokinetics of the response to the local delivery of nomifensine. Overall, the findings reported here confirm the benefits arising from the ability to monitor dopamine via microdialysis at high sensitivity and at high temporal resolution.

An integrated approach for profiling oxidative metabolites and glutathione adducts using liquid chromatography coupled with ultraviolet detection and triple quadrupole-linear ion trap mass spectrometry.

The use of liquid chromatography (LC) coupled with triple quadrupole linear ion trap (Qtrap) mass spectrometry (MS) for both quantitative and qualitative analysis in drug metabolism and pharmacokinetic studies is of great interest. Here, a new Qtrap-based analytical methodology for simultaneous detection, structural characterization and semi-quantitation of in vitro oxidative metabolites and glutathione trapped reactive metabolites was reported. In the current study, combined multiple ion monitoring and multiple reaction monitoring were served as surveying scans to trigger product ion spectral acquisition of oxidative metabolites and glutathione adduct, respectively. Then, detection of metabolites and recovery of their MS/MS spectra were accomplished using multiple data mining approaches. Additionally, on-line ultraviolet (UV) detection was employed to determine relative concentrations of major metabolites. Analyses of metabolites of clozapine and nomifensine in rat liver microsomes not only revealed multiple oxidative metabolites and glutathione adducts, but also identified their major oxidative metabolism and bioactivation pathways. The results demonstrated that the LC/UV/MS method enabled Qtrap to perform the comprehensive profiling of oxidative metabolites and glutathione adducts in vitro.

Enhanced Dopamine Release by Dopamine Transport Inhibitors Described by a Restricted Diffusion Model and Fast-Scan Cyclic Voltammetry.

Fast-scan cyclic voltammetry (FSCV) using carbon fiber electrodes is widely used to rapidly monitor changes in dopamine (DA) levels in vitro and in vivo. Current analytical approaches utilize parameters such as peak oxidation current amplitude and decay times to estimate release and uptake processes, respectively. However, peak amplitude changes are often observed with uptake inhibitors, thereby confounding the interpretation of these parameters. To overcome this limitation, we demonstrate that a simple five-parameter, two-compartment model mathematically describes DA signals as a balance of release (r/ke) and uptake (ku), summed with adsorption (kads and kdes) of DA to the carbon electrode surface. Using nonlinear regression, we demonstrate that our model precisely describes measured DA signals obtained in brain slice recordings. The parameters extracted from these curves were then validated using pharmacological manipulations that selectively alter vesicular release or DA transporter (DAT)-mediated uptake. Manipulation of DA release through altering the Ca(2+)/Mg(2+) ratio or adding tetrodotoxin reduced the release parameter with no effect on the uptake parameter. DAT inhibitors methylenedioxypyrovalerone, cocaine, and nomifensine significantly reduced uptake and increased vesicular DA release. In contrast, a low concentration of amphetamine reduced uptake but had no effect on DA release. Finally, the kappa opioid receptor agonist U50,488 significantly reduced vesicular DA release but had no effect on uptake. Together, these data demonstrate a novel analytical approach to distinguish the effects of manipulations on DA release or uptake that can be used to interpret FSCV data.

Alternative pharmacological strategies for adult ADHD treatment: a systematic review.

Adult Attention Deficit Hyperactivity Disorder (ADHD) is a prevalent psychiatric condition associated with high disability and frequent comorbidity. Current standard pharmacotherapy (methylphenidate and atomoxetine) improves ADHD symptoms in the short-term, but poor data were published about long-term treatment. In addition a number of patients present partial or no response to methylphenidate and atomoxetine. Research into the main database sources has been conducted to obtain an overview of alternative pharmacological approaches in adult ADHD patients. Among alternative compounds, amphetamines (mixed amphetamine salts and lisdexamfetamine) have the most robust evidence of efficacy, but they may be associated with serious side effects (e.g. psychotic symptoms or hypertension). Antidepressants, particularly those acting as noradrenaline or dopamine enhancers, have evidence of efficacy, but they should be avoided in patients with comorbid bipolar disorder. Finally metadoxine and lithium may be particularly suitable in case of comorbid alcohol misuse or bipolar disorder.

Modeling the kinetic diversity of dopamine in the dorsal striatum.

Dopamine is an important neurotransmitter that exhibits numerous functions in the healthy, injured, and diseased brain. Fast scan cyclic voltammetry paired with electrical stimulation of dopamine axons is a popular and powerful method for investigating the dynamics of dopamine in the extracellular space. Evidence now suggests that the heterogeneity of electrically evoked dopamine responses reflects the inherent kinetic diversity of dopamine systems, which might contribute to their diversity of physiological function. Dopamine measurements by fast scan cyclic voltammetry are affected by the adsorption of dopamine to carbon fiber electrodes. The temporal distortion caused by dopamine adsorption is correctable by a straightforward mathematical procedure. The corrected responses exhibit excellent agreement with a dopamine kinetic model cast to provide a generic description of restricted diffusion, short-term plasticity of dopamine release, and first-order dopamine clearance. The new DA kinetic model brings to light the rich kinetic information content of electrically evoked dopamine responses recorded via fast scan cyclic voltammetry in the rat dorsal striatum.

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.