Some characteristics of rat nigrostriatal dopaminergic neuroconnection during development--a combined immunocytochemical and electron-microscopic study

The characteristics of the nigrostriatal dopaminergic neuroconnections in developing rats are studied by combined immunocytochemical and electron-microscopic techniques, with an antibody to tyrosine hydroxylase (TH). From the embryonic day 21 on, some of the TH-positive nerve fibers are densely packed in the striatum to form a patch-like dopamine island. The percentage of the TH-positive nerve terminals among the labeled profiles is much higher inside than outside the dopamine island (P less than 0.01). On the other hand, the TH-positive terminals mainly form symmetrical axon-dendritic synapses, while most of the TH-negative terminals form asymmetrical axon-spinous synapses. The functional significance of the characterized dopaminergic connection is discussed.     

SOME CHARACTERISTICS OF RAT NIGROSTRIATAL DOPAMINERGIC NEUROCONNECTION DURING DEVELOPMENT——A COMBINED IMMUNOCYTOCHEMICAL AND ELECTRON-MICROSCOPIC STUDY

The characteristics of the nigrostriatal dopaminergic neuroconnections in developing rats are studied by combined immunocytochemical and electron-microscopic techniques, with an antibody to tyrosine hydroxylase (TH). From the embryonic day 21 on, some of the THpositive nerve fibers are densely packed in the striatum to form a patch-like dopamine island. The percentage of the TH-positive nerve terminals among the labeled profiles is much higher inside than outside the dopamine island (P<0.01). On the other hand, the TH-positive terminals mainly form symmetrical axon-dendritic synapses, while most of the TH-negative terminals form asymmetrical axon-spinous synapses. The functional significance of the characterized dopaminergic connection is discussed.     

Myoglobin modification by enzyme-generated dopamine reactive species

The generation of reactive quinone species (DAQ) from oxidation of dopamine (DA) is involved in neurodegenerative pathologies like Parkinson's disease (A. Borta, G. U. H?glinger, J. Neurochem. 2007, 100, 587-595). The oxidation of DA to DAQ can occur either in a single two-electron process or in two consecutive one-electron steps, through semiquinone radicals, giving rise to different patterns of reactions. The former type of reaction can be promoted by tyrosinase, the latter by peroxidases in the presence of H(2)O(2), which can be formed under oxidative stress conditions. Both enzymes were employed for the characterization of the thiol-catechol adducts formed by reaction of DA and cysteine or glutathione, and for the identification of specific amino acid residues modified by DAQs in two representative target proteins, human and horse heart myoglobin. Our results indicate that the cysteinyl-DA adducts are formed from the same quinone intermediate independently of the mechanism of DA oxidation, and that the hallmark of a radical mechanism is the formation of the cystine dimer. The reactivity of quinone species also controls the DA-promoted derivatization of histidine residues in proteins. However, for the modification of the cysteine residue in human myoglobin, a radical intramolecular mechanism has been proposed, in which the protein acts both as the catalyst and target of the reaction. Most importantly, the modification of myoglobins through DAQ linkages, and in particular by DA oligomers, has dramatic effects on their stability, as it induces protein unfolding and incorporation into insoluble melanic precipitates.     

Stress and the dopaminergic reward system

Dopamine regulates reward-related behavior through the mesolimbic dopaminergic pathway. Stress affects dopamine levels and dopaminergic neuronal activity in the mesolimbic dopamine system. Changes in mesolimbic dopaminergic neurotransmission are important for coping with stress, as they allow adaption to behavioral responses to various environmental stimuli. Upon stress exposure, modulation of the dopaminergic reward system is necessary for monitoring and selecting the optimal process for coping with stressful situations. Aversive stressful events may negatively regulate the dopaminergic reward system, perturbing reward sensitivity, which is closely associated with chronic stress-induced depression. The mesolimbic dopamine system is excited not only by reward but also by aversive stressful stimuli, which adds further intriguing complexity to the relationship between stress and the reward system. This review focuses on lines of evidence related to how stress, especially chronic stress, affects the mesolimbic dopamine system, and discusses the role of the dopaminergic reward system in chronic stress-induced depression.Subject terms: Molecular neuroscience, Motivation     

Simultaneous determination of norepinephrine, dopamine, 5-hydroxytryptamine and their main metabolites in rat brain using high-performance liquid chromatography with electrochemical detection. Enzymatic hydrolysis of metabolites prior to chromatography

In order to measure turnover rates of the noradrenergic, dopaminergic, and serotonergic transmitter systems in rat brain, a method was developed by which norepinephrine, dopamine, and 5-hydroxytryptamine, and their main metabolites 3-methoxy-4-hydroxyphenylglycol, 3,4-dihydroxyphenylacetic acid, 3-methoxy-4-hydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid, could be measured simultaneously. High-performance liquid chromatography in the reversed-phase mode, including ion pairing, separated the transmitters and their metabolites well. By means of enzymatic hydrolysis of the sample prior to chromatography, it was also possible to measure the conjugated forms of the metabolites. Since there was no prepurification step, the hydrolysed supernatants of tissue homogenates were injected directly into the chromatographic system; additional selectivity tests were necessary. Peak identification was confirmed by comparison of hydrodynamic voltagrams and capacity factors at different pH values of the mobile phase of the components in the sample and the standard solution. The method is demonstrated by analysing mediobasal hypothalamic tissues of probenecid-treated rats.     

Detection of dopamine in the presence of excess ascorbic acid at physiological concentrations through redox cycling at an unmodified microelectrode array

The electrochemical behavior of dopamine was examined under redox cycling conditions in the presence and absence of a high concentration of the interferent ascorbic acid at a coplanar, microelectrode array where the area of the generator electrodes was larger than that of the collector electrodes. Redox cycling converts a redox species between its oxidized and reduced forms by application of suitable potentials on a set of closely located generator and collector electrodes. It allows signal amplification and discrimination between species that undergo reversible and irreversible electron transfer. Microfabrication was used to produce 18 individually addressable, 4-μm-wide gold band electrodes, 2 mm long, contained in an array having an interelectrode spacing of 4 μm. Because the array electrodes are individually addressable, each can be selectively biased to produce an overall optimal electrochemical response. Four adjacent microbands were shorted together to serve as the collector, and were flanked on each side by seven microbands shorted as the generator (a ratio of 1:3.5 of electroactive area, respectively). This configuration achieved a detection limit of 0.454?±?0.026 μM dopamine at the collector in the presence of 100 μM ascorbic acid in artificial cerebrospinal fluid buffer, concentrations that are consistent with physiological levels. Enhancement by surface modification of the microelectrode array to achieve this detection limit was unnecessary. The results suggest that the redox cycling method may be suitable for in vivo quantification of transients and basal levels of dopamine in the brain without background subtraction.

A modified analytical method for total antioxidant potential assay using RP-HPLC with electrochemical detection and its application for pro- and antioxidative properties of dopamine measurement

Total antioxidant potential (TAP) is usually measured using photometric or fluorometric assays. Preliminary results of a reversed-phase high-performance liquid chromatography--electrochemical detection assay are given. The method is based on the generation of hydroxyl radicals in a Fenton reaction and analysis of the product of their interaction with p-hydroxybenzoic acid (3,4-dihydroxybenzoic acid). The method is applied to estimate the TAP of dopamine. As a result, depending on the concentration, dopamine is pro- or antioxidant. The results are compared with TAP measurements using a standard photometric method.     

Liquid chromatographic studies on the aqueous solution conformation of substituted benzamide drug models

The aqueous solution conformation of a series of model benzamides related to the orthopramide dopamine receptor antagonists is evaluated by reversed-phase liquid chromatography (LC). The structure-retention relationship studies demonstrate the existence of an intramolecular hydrogen bond formed in aqueous solution for these compounds. The six-membered pseudoring formed by the association of the amide N-H and the oxygen of the 2-methoxy group (N-H...O) produces enhanced reversed-phase retention (increased capacity factors, k') relative to the 3- and 4-isomers. Deletion of either the acceptor oxygen or the donor N-H results in decreased C18 retention of the 2-isomer relative to the 3- and 4-isomers. These structure-retention relationship studies reveal the value of reversed-phase LC methods for evaluation of the aqueous solution conformation of the orthopramide-type compounds. Further studies show that N-substituted 2-hydroxybenzamides are intramolecularly associated in aqueous solution through either N-H...O or O-H...O six-membered rings. The 6-methoxysalicylamides are believed to be stabilized through O-H to carbonyl oxygen bonding in addition to the N-H...O pseudoring. For the 2,6-dimethoxybenzamides, steric factors prevent methoxy-amide coplanarity, thus no intramolecular hydrogen bond is formed. The result is a dramatic decrease in retention for the 2,6-isomer relative to the other positional isomers. These studies suggest that remoxipride and related 2,6-dimethoxybenzamides cannot form the six-membered pseudoring believed to be topographically equivalent to the aromatic ring in dopamine.     

Assay for dopamine beta-hydroxylase in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection

A highly sensitive assay for dopamine beta-hydroxylase activity in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection is described. Norepinephrine, formed enzymatically from the substrate dopamine, and epinephrine (internal standard), after clean-up with a cation-exchange cartridge (Toyopak IC-SP M), are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-80TM. The detection limit for norepinephrine formed enzymatically is 5 pmol per assay tube.     

多巴胺D2受体显像剂125I-DMAIBZM的合成及生物分布实验

为提高苯甲酰胺类衍生物（S）-N-（1-乙基-2-吡咯烷基）甲基-4-氨基-2-甲氧基苯酰胺（ABZM）的入脑量,对其结构进行改造,得到了新的化合物（S）-4-二甲氨基-N-（1-乙基-2-吡咯烷基）甲基-2-甲氧基苯酰胺（DMABZM）.通过Idogen法对DMABZM进行标记得到标记化合物125I-DMAIBZM,标记率为74%,放化纯度达到99%． 体外放射配基结合实验测得DMABZM的IC50为2.9589×10-7 mol?L-1,表明它与能与多巴胺D2受体特异性结合且具有较高的亲和力,在小鼠体内的分布实验中,该标记物纹状体/小脑的比值可达6.5左右,说明了该标记化合物与纹状体的结合有较高的特异性和亲和力．125I-DMAIBZM的脂溶性显著大于125I-AIBZM,入脑量有较大的提高．结论：125I（123I）-DMAIBZM有望用于多巴胺D2受体的显像．     

Direct determination of vanillylmandelic acid in human urine by reversed-phase HPLC

A reversed-phase HPLC method for the determination of epinephrine, norepinephrine, dopamine, and vanillylmandelic acid (VMA) has been developed. The concentration of VMA in the urine of hypertensive patients was measured by direct injection after centrifugation. The method is useful for the diagnosis of pheochromocytoma.     

Preparation of dopamine 3-O-sulphate and dopamine 4-O-sulphate as reference substances and high-performance liquid chromatographic trace determination

Simple syntheses of the biologically important but hitherto difficult to obtain dopamine sulpho conjugates dopamine 3-O-sulphate (I) and dopamine 4-O-sulphate (II), as analytical reference substances, starting from dopamine hydrochloride are described. A method for the determination of I and II with reversed-phase high-performance liquid chromatographic separation and coulometric detection in human urine together with calibration and current-voltage curves are presented. Detection limits of approximately 100 pg of I or II and unequivocal substance identifications even in very complex substrates such as human urine are reported.     

Oxidation of dopamine on multi-walled carbon nanotubes

Novel films consisting of multi-walled carbon nanotubes (MWCNTs) were fabricated by means of the chemical vapor deposition technique with decomposition of either acetonitrile (ACN) or benzene (BZ) in the presence of ferrocene (FeCp₂) which served as catalyst. The electrochemical response of the two different kinds of MWCNT-based films, further referred to as MWCNT-ACN and MWCNT-BZ, towards the oxidation of dopamine (DA) to dopamine-o-quinone (DAQ) was tested by means of cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. Both MWCNT-based films exhibit quasi-reversible response towards DA/DAQ with some slight kinetic differences; specifically, the charge-transfer process was found to be faster on MWCNT-ACN (k _s?=?35.3?×?10^?3 cm s^?1) compared to MWCNT-BZ (k _s?=?6.55?×?10^?3 cm s^?1). The detection limit of MWCNT-BZ for DA (0.30 μM) appears to be poorer compared to that of MWCNT-ACN (0.03 μM), but nevertheless, both MWCNT-based films exhibit greater detection ability compared to other electrodes reported in the literature. The sensitivities of MWCNT-ACN and MWCNT-BZ towards DA/DAQ were determined as 0.65 and 0.22?A M^?1 cm^?2, respectively. The findings suggest that the fabricated MWCNT-based electrodes can be successfully applied for the detection of molecules with biological interest.     

Application of fast LC to pharmaceutical analysis: dopamine.HCl

A reversed-phase HPLC method for the determination of the dopamine hydrochloride content of pharmaceutical intravenous solutions is developed using short (3-cm) columns packed with 3-micron particles. Total analysis time for the stability indicating assay, which is capable of separating the analyte from all its common degradates and formulation matrix components, is 2 min. The assay is equivalent in performance (accuracy, precision, separation behavior, ruggedness) to a standard method recommended by the United States Pharmacopeia (USP). Common components of conventional HPLC systems are evaluated with respect to their utility for this application of fast LC.     

The preparation of dopaminergic agonist [5,8-3H] (+/-) 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene at high specific activity

The rigid dopamine analogue (+/-) 6,7-ADTN was originally synthesized to probe the geometrical constraints of the dopamine receptor family and [³H] (+/-) 6,7-ADTN was required for receptor binding assay. The radioligands was prepared by means of catalytic tritium dehalogenation of a suitable dibromo precursor and characterized by HPLC and tritium NMR. It has proven very useful as a tool to study dopaminergic receptors.     

Computer-aided molecular modeling of a D2-agonist dopamine pharmacophore

Summary Using computer-aided molecular modeling techniques to analyze models recently proposed for the receptor binding sites of dopaminergic agonists, we superimposed the chemical structures of various compounds that mimic the pharmacological behavior of dopamine, as well as inactive enantiomers, on a postulated three-dimensional frame of reference. We analyzed the vector directionalities of the lone pairs of the nitrogen common to these molecules, and the acidic hydrogen of phenols (in aminoindanes, aminotetralins, apomorphines,p-phenol-piperazines, octahydrobenzo(g)quinolines, octahydrobenzo(f)quinolines, and benzazepines) or of nitrogen (in ergoline-type compounds and related structures). This model, when expressed as distances from that of the reference compound pergolide, correlates with the dopaminergic binding affinity observed in compounds previously reported to act on the dopaminergic system in the central nervous system (CNS). The regression analysis of log K_D with respect to the distances of the vectors of the acidic hydrogen support the hypothesis that these compounds bind to the receptor as donors in hydrogen bond formation.     

Selective solid-phase extraction of catecholamines by the chemically modified polymeric adsorbents with crown ether

A simple and selective one-step solid-phase extraction procedure using chemically modified polymer resin (Amberlite XAD-4) with crown ether was investigated for the measurement of urinary catecholamines. After loading the urine samples (adjusted to pH 4) on the synthesized adsorbent cartridge, the column was washed with methanol followed by water and then the adsorbed catecholamines were eluted by 1.0 mL of 6.0 M acetic acid. The effectiveness of sample clean-up method was demonstrated by reversed-phase ion-pair high-performance liquid chromatography with electrochemical detection. Under optimal condition, the recoveries of epinephrine, norepinephrine, and dopamine from spiked urine sample were >86% for all catecholamines. The detection limits (n=5) for epinephrine, norepinephrine, and dopamine were 37, 52, and 46 nmol/L, respectively.     

Determination of enantiomeric purity of the new D-2 dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437) by reversed-phase high-performance liquid chromatography after pre-column derivatization with D(+)-glucuronic acid

This paper describes an enzymic derivatization procedure that allows accurate determination of very small amounts of enantiomeric impurities in the D-2 dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437). After pre-column glucuronidation of the individual enantiomers, two diastereoisomers were formed which were separated by reversed-phase high-performance liquid chromatography. An enantiomeric purity of 99.84% was calculated for the (-)-enantiomer, against 99.89% for the (+)-enantiomer. The assay was validated by spiking 1% of the (-)-enantiomer in the (+)-enantiomer. A high accuracy (error 4.5%) and precision (coefficient of variation 2.9%, n = 5) of the method were established.     

In vivo imaging of brain dopaminergic neurotransmission system in small animals with high-resolution single photon emission computed tomography.

High-resolution single photon emission computed tomography (SPECT) provides a unique capability to image the biodistribution of radiolabeled molecules in small laboratory animals. Thus, we applied the high-resolution SPECT to in vivo imaging of the brain dopaminergic neurotransmission system in common marmosets using two radiolabeled ligands, [123I]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane (beta-CIT) as a dopamine transporter (DAT) ligand and [123I]iodobenzamide (IBZM) as a dopamine D2 receptor (D2R) ligand. Specific images of the striatum, a region with a high density of dopaminergic synapses, were obtained at 240 min and 60 min after injection of [123I]beta-CIT and [123I]IBZM, respectively. Furthermore, a significantly low accumulation of [123I]beta-CIT in the striatum was observed in MPTP-treated animals compared with results for a control group, and a similar accumulation in the control group was observed with the pretreatment of deprenyl in the MPTP-treated animals. However, the striatal accumulation of [123I]IBZM showed no changes among the control, MPTP-treated, and deprenyl-MPTP-treated groups. These SPECT imaging results agreed well with those of DA concentration and motor behavior. Since MPTP destroys nigrostriatal dopamine nerves and produces irreversible neurodegeneration associated with Parkinsonian syndrome, SPECT imaging data in this study demonstrated that deprenyl shows its neuroprotective effect on Parkinsonism by protecting against the destruction of presynaptic dopamine neurons.     

The effect of N‐acetylcysteine on amphetamine‐mediated dopamine release in rat brain striatal slices by ion‐pair reversed‐phase high performance liquid chromatography

The amphetamine (AMPH)‐induced alteration in rat brain dopamine levels modified by N‐acetylcysteine (NAC) administration has been examined using isocratic ion‐pair reversed‐phase high‐performance liquid chromatography with electrochemical detection. The aim of the development of a novel validated evaluation scheme implying a double AMPH challenge was to enhance the efficiency of AMPH‐triggered dopamine release measurements in rat brain striatal slices by improving the reproducibility of the results. The proposed experimental protocol was tested in vivo and proved to be capable of fast and reliable drug screening for tracing the effect of NAC as a model compound in AMPH‐mediated dopaminergic response. The subcellular localization of the dopamine mobilizing effect of NAC has been established indirectly by the use of an irreversible dopamine vesicular depletor, reserpine. The antioxidant NAC at 10 mm plays an important role in the complete suppression of acute AMPH‐elicited dopamine release. The possible role of this quenching effect is discussed. Copyright © 2009 John Wiley & Sons, Ltd.