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In order to make high-performance liquid chromatography (HPLC) more widely available at home and in small-scale settings, we have simplified two of its most costly modules, namely the pump and the detector. This should make the setup affordable for home or small laboratory use. A manual HPLC pump was constructed so as to fit into a caulk gun from a local hardware store enabling the generation of 100-150 bar of pressure. In order to limit the pressure drop during the running of a chromatogram, a pulse dampener was developed. We further modified the electrochemical detection (ECD) system so as to use a cheap boron-doped diamond electrode with an overlay of thin filter paper, causing an eluent flow over the electrode by wicking and gravity. Both the pump and the detector are at least ten times cheaper than conventional HPLC modules. Using a home-packed Jupiter Ⓡ Proteo reversed phase capillary column we show how this low-cost HPLC system generates well resolving chromatograms after direct injection of fresh urine. The ECD did not lose its sensitivity during regular use over more than half a year. For homovanillic acid (HVA), which is of medical interest, we measured a linear dynamic range of two orders of magnitude, a detection limit of HVA in the injected sample of 3 μM and a coefficient of variation <10%. The contribution to peak broadening by the detector was much smaller than the contributions by the injector and by the column. After consumption of table olives containing hydroxytyrosol (HT), its metabolite HVA in the corresponding urine could be measured quantitatively. An approach to quantify HT in table olives is presented, as well. This method provides a new tool for investigating physiology of oneself or of dear ones at home., Competing Interests: Declaration of Competing Interest The authors declare that JL and PvdS are board members of a non-profit Foundation for the promotion of the use of chromatography at home., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology is a growing research field which provides valuable information on community drug use and chemical exposure. One parameter critical to estimations of drug use is the catchment area population. A population biomarker could be used to provide this information. This study evaluated the analytical suitability of three endogenous biomarkers of human activity: the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) which has previously been proposed and two further candidates, the catecholamine metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA). An analytical method involving derivatization was developed and validated for two candidates, 5-HIAA and HVA by liquid chromatography - mass spectrometry. The best performance was obtained for VMA as the underivatized analyte. The derivatized extracts produced a 100 times better sensitivity. The three neurotransmitter metabolites were evaluated as population biomarkers in wastewater samples. All were stable in sample, not lost upon filtration and showed stable inter-day mass loads over seven days for a metropolitan wastewater treatment plant. When applied to a small community during a festival period, mass loads of both HVA and VMA reflected the increase in the catchment population, whilst 5-HIAA proved to be more variable., Competing Interests: Declaration of Competing Interest All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for important intellectual content; and (c) approval of the final version. This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript, (Copyright © 2019 Elsevier B.V. All rights reserved.)

A new method for the simultaneous determination of two tumour biomarkers, homovanillic (HVA) and vanillylmandelic acid (VMA), using flow injection analysis (FIA) with amperometric detection (AD) at a commercially available boron doped diamond electrode (BDDE) was developed. It was found that this method is suitable for the determination of HVA (in the presence of VMA) and VMA (in the presence of HVA) in optimum medium of Britton-Robinson buffer (0.04 mol L -1 , pH 3.0). Calibration dependences consist of two linear parts for both biomarkers, the first one being in the concentration range from 1 to 10 μmol L -1 and the second one from 10 to 100 μmol L -1 (with obtained LODs 0.44 μmol L -1 for HVA and 0.34 μmol L -1 for VMA, respectively). To minimize any negative effects related to the passivation of the working electrode, suitable cleaning pulses (+2.4 V for 30 s) were imposed on the working electrode after each measurement. An attempt to use FIA with multiple pulse amperometric detection to determine both analytes in one run was not successful. Changing potentials in short intervals in multiple pulse detection probably results in mutual interaction of analytes and/or products of their electrochemical oxidation, thus preventing the application of this approach., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Camel milk is reported as anti-diabetic, hepato-protective, anticancer, antioxidant, antiviral and neuroprotectant in numerous studies. Based on its neuroprotective profile, camel milk is investigated for its possible beneficial effect in treating anxiety and depression and its effect on brain biogenic amines in the present study. Head dip, cage crossing, stationary rod, elevated plus-maze, open field, light & dark box and forced swim tests were used to measure change in rodents' behavior after camel milk administration. Any possible change in brain biogenic amines level after camel milk treatment was evaluated using High Performance Liquid Chromatography (HPLC) technique. Camel milk administration resulted in significant increase (p<0.001) in exploratory and locomotor activity and showing anxiolytic behavior in rodents. In depression-like model, rats showed significant increase (p<0.001) in struggling time after 30-days administration of camel milk. HPLC detection of brain biogenic amines revealed significant increase (p<0.001) in norepinephrine, insignificant increase in 5-hydroxytryptamine and significant decrease (p<0.001) in dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid in camel milk treated group. Based on above findings, camel milk is suggested as anxiolytic and antidepressant in the administered doses. However, further experimental and clinical investigations are required to authenticate the same at different doses.

Background and objectives: Melanin, which has a confirmed role in melanoma cell behaviour, is formed in the process of melanogenesis and is synthesized from tryptophan, L-tyrosine and their metabolites. All these metabolites are easily detectable by chromatography in urine. Materials and Methods: Urine samples of 133 individuals (82 malignant melanoma patients and 51 healthy controls) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC). The diagnosis of malignant melanoma was confirmed histologically. Results: Chromatograms of melanoma patients showed increased levels of 5,6-dihydroxyindole-2-carboxylic acid, vanilmandelic acid, homovanilic acid, tryptophan, 5-hydroxyindole-3-acetic acid, and indoxyl sulphate compared to healthy controls. Concentration of indoxyl sulphate, homovanilic acid and tryptophan were significantly increased even in the low clinical stage 0 of the disease (indoxyl sulphate, homovanilic acid and tryptophan in patients with clinical stage 0 vs. controls expressed as medium/ interquartile range in µmol/mmol creatinine: 28.37/15.30 vs. 5.00/6.91; 47.97/33.08 vs. 7.33/21.25; and 16.38/15.98 vs. 3.46/6.22, respectively). Conclusions: HPLC detection of metabolites of L-tyrosine and tryptophan in the urine of melanoma patients may play a significant role in diagnostics as well as a therapeutic strategy of melanoma cancer.

In this study, a simultaneous assay for catecholamines and their metabolites in the brain was established using liquid chromatography-mass spectrometry (LC-MS). To achieve complete separation, a cation-exchange/reversed-phase mixed-mode copolymer resin column containing 0.81 wt% sulfo groups was used for the simultaneous LC-MS assay. The analyzed catecholamines were dopamine (DA), norepinephrine (NE), and epinephrine (E), while the metabolites lacking amino groups were 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol (MHPG). The metabolites were separated and detected using LC-MS, on columns with and without sulfo groups. However, we could not achieve adequate separation of catecholamines on both columns using a gradient elution of 0 - 50 (v/v)% methanol containing 0.1 (v/v)% formic acid (FA). When volatile ion-pairing reagents were added to the mobile phase, they improved the retention and detection of catecholamines on the sulfonated mixed-mode column. Under optimized elution conditions, which involved a linear gradient elution of water containing 0.1 (v/v)% FA to 50 (v/v)% acetonitrile in 50 mM ammonium formate at 40°C and a 0.20 mL/min rate, all six target molecules were simultaneously detected within 25 min, when using negative mode LC-MS on a sulfonated mixed-mode column. The limits of detection (LODs) for DA, NE, E, DOPCA, HVA, and MHPG were determined to be 20.7, 12.6, 74.6, 1110, 18.7, and 3196 nM, respectively. Moreover, the established LC-MS assay allowed the detection of endogenous DA, NE, and HVA, in normal mouse brain samples at concentrations higher than 20, 9, and 4 pmol/mg, respectively.

Two novel phenanthrenoids, juncuenin H (1) and dijuncuenin B (2), together with eight known phenanthrenoids, effusol (3), dehydroeffusol (4), juncusol (5), dehydrojuncusol (6), juncuenin B (7), dehydrojuncuenin B (8), juncuenin A (9), and dehydrojuncuenin A (10), were isolated from the underground parts of Juncus setchuenensis. The structures of the compounds were determined by 1D and 2D NMR and mass spectroscopy. The anxiolytic activities of compounds 1, 6, 9, and 10 were evaluated. In order to explore the mechanisms underlying their anxiolytic activities, the levels of serotonin (5-HT), dopamine (DA), and their metabolites in the cerebral cortex and hippocampus of mice treated with compound 1 were determined by quantitative mass spectrometry. The mice treated with compound 1 had significantly lower levels of 5-HT, 3-methoxytyramine (3-MT), 5-hydroxyindole-3-acetic acid (5-HIAA), homovanillic acid (HVA), and 3, 4-dihydroxyphenylacetic acid (DOPAC) in the cerebral cortex than those of the vehicle control-treated mice. The levels of HVA and 5-HIAA in the hippocampus were also significantly lower in the mice treated with compound 1 than in the control group mice. These results suggest that the metabolic changes, reflected in the levels of DA and/or 5-HT, may contribute to the anxiolytic activity of the phenanthrenoids studied herein., (Copyright © 2019. Published by Elsevier B.V.)

The experiments were performed in male albino outbred mice kept in a group and under the conditions of long-term social isolation. The changes in the monoaminergic systems of the left and right hemispheres of the brain after acute hypoxia with hypercapnia have been studied. The levels of dopamine (DA), serotonin (5-HT) and their metabolites - dioxyphenylacetic (DOPAC), homovanillic (HVA), and 5-hydroxyindoleacetic (5-HIAA) acids - were determined by HPLC in the cerebral cortex, hippocampus and striatum of the right and left sides of the brain. In the control mice kept both in the group and under the conditions of social isolation, a higher content of DA in the cortex of the left hemisphere has been found. In the other brain structures the monoamine content was symmetric. In the cerebral cortex of the mice in the group, acute hypoxia with hypercapnia led to a right-sided increase in the DA and 5HT levels. At the same time, the DOPAC content decreased in the left cortex. In mice in the group, under the hypoxia with hypercapnia conditions, the DA level in the left hippocampus increased. In the striatum, the content of monoamines and their metabolites did not change significantly. In animals kept for a long time under the conditions of social isolation, hypoxia with hypercapnia no statistically significant changes in the monoamines and their metabolites levels were found. It has been concluded that the preliminary maintenance under the conditions of prolonged social isolation changes the reaction of central monoaminergic systems to acute hypoxia with hypercapnia.

Objective To investigate the role of S100B in the development of Parkinson's disease (PD) and explore the possibility of brain-specific S100B transgenic mice as PD animal model. Methods The hS100B transgenic mice were established. The mice were divided into S100B transgenic group (TG),S100B knockout group (KG),and the non-transgenic control group (CG). Motor coordination ability of mice was measured by the rota-rod and pole-climbing test. The expressions of S100B,dopamine D1 receptor,dopamine D2 receptor,G protein-coupled receptor kinase (GRK)2,GRK5,and tyrosine hydroxylase in brain tissue were detected by reverse transcription-polymerase chain reaction and Western blot. The levels of tyrosine,levodopa,dopamine,and homovanillic acid in brain tissue were measured by high-performance liquid chromatography coupled with fluorescence detection. Results Compared with CG,the S100B protein expression in brain tissue significantly increased in TG (P<0.05);the motor coordination ability of mice showed progressive decline (P<0.05);the mRNA and protein expressions of dopamine D2 receptor and GRK2 significantly decreased (P<0.05);the levels of levodopa,dopamine,and homovanillic acid were significantly elevated (P<0.05);the expression of tyrosine hydroxylase was also down-regulated,although there was no significant difference (P>0.05). Compared with CG,there was no obvious change of the above indicators in KG (all P>0.05). Conclusion S100B plays an important role in the motor coordination abnormity of PD. The brain-specific S100B transgenic mice can be used in research on the role of S100B gene in the development of PD.

Neuroblastoma is a pediatric neuroblastic tumor arising in the sympathetic nervous crest cells. A high grade of Neuroblastoma is characterized by a high urinary excretion of homovanillic acid and dopamine. In this work l-leucine modified Sol-Gel-Carbon electrode was used for a sensitive voltammetric determination of homovanillic acid and dopamine in urine. The electrochemical response characteristics were investigated by cyclic and differential pulse voltammetry; the modified electrode has shown an increase in the effective area of up to 40%, a well-separated oxidation peaks and an excellent electrocatalytic activity. High sensitivity and selectivity in the linear range of 0,4-100μML -1 of homovanillic acid and 10-120μML -1 of dopamine were also obtained. Moreover, a sub-micromolar limit of detection of 0.1μM for homovanillic acid and 1.0μM for the dopamine was achieved. Indeed, high reproducibility with simple preparation and regeneration of the electrode surface made this electrode very suitable for the determination of homovanillic acid and dopamine in pharmaceutical and clinical preparations. The mechanism of homovanillic acid and the electrochemical oxidation at l-leucine modified Sol-Gel-Carbon electrode is described out the B3P86/6-31+G(d,p) level of theory as implemented in Gaussian software., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Neuroblastoma is the most common extracranial solid tumour occurring in childhood and has a diverse clinical presentation and course depending on the tumour biology. Unique features of these neuroendocrine tumours are the early age of onset, the high frequency of metastatic disease at diagnosis and the tendency for spontaneous regression of tumours in infancy. The most malignant tumours have amplification of the MYCN oncogene (encoding a transcription factor), which is usually associated with poor survival, even in localized disease. Although transgenic mouse models have shown that MYCN overexpression can be a tumour-initiating factor, many other cooperating genes and tumour suppressor genes are still under investigation and might also have a role in tumour development. Segmental chromosome alterations are frequent in neuroblastoma and are associated with worse outcome. The rare familial neuroblastomas are usually associated with germline mutations in ALK, which is mutated in 10-15% of primary tumours, and provides a potential therapeutic target. Risk-stratified therapy has facilitated the reduction of therapy for children with low-risk and intermediate-risk disease. Advances in therapy for patients with high-risk disease include intensive induction chemotherapy and myeloablative chemotherapy, followed by the treatment of minimal residual disease using differentiation therapy and immunotherapy; these have improved 5-year overall survival to 50%. Currently, new approaches targeting the noradrenaline transporter, genetic pathways and the tumour microenvironment hold promise for further improvements in survival and long-term quality of life.

A major hypothesis in addiction research is that alcohol induces neuroadaptations in the mesolimbic dopamine (DA) system and that these neuroadaptations represent a key neurochemical event in compulsive drug use and relapse. Whether these neuroadaptations lead to a hypo- or hyperdopaminergic state during abstinence is a long-standing, unresolved debate among addiction researchers. The answer is of critical importance for understanding the neurobiological mechanism of addictive behavior. Here we set out to study systematically the neuroadaptive changes in the DA system during the addiction cycle in alcohol-dependent patients and rats. In postmortem brain samples from human alcoholics we found a strong down-regulation of the D1 receptor- and DA transporter (DAT)-binding sites, but D2-like receptor binding was unaffected. To gain insight into the time course of these neuroadaptations, we compared the human data with that from alcohol-dependent rats at several time points during abstinence. We found a dynamic regulation of D1 and DAT during 3 wk of abstinence. After the third week the rat data mirrored our human data. This time point was characterized by elevated extracellular DA levels, lack of synaptic response to D1 stimulation, and augmented motor activity. Further functional evidence is given by a genetic rat model for hyperdopaminergia that resembles a phenocopy of alcohol-dependent rats during protracted abstinence. In summary, we provide a new dynamic model of abstinence-related changes in the striatal DA system; in this model a hyperdopaminergic state during protracted abstinence is associated with vulnerability for relapse.

Background: Sepsis-associated encephalopathy (SAE) is defined as a diffuse or multifocal cerebral dysfunction that generally occurs early during severe sepsis. The complete pathophysiology of SAE is unknown, but several mechanisms including endotoxins, inflammatory mediators, the alteration of amino acids and of neurotransmitters, apoptosis, oxidative stress, and blood-brain barrier dysfunction have been suggested. The aim of the present study was to explore the relationship between behavioral stereotypy and plasma levels of tumor necrosis factor-alpha (TNF-α) and malondialdehyde (a marker of lipid peroxidation), and brain homovanillic acid content (a marker of dopamine turnover) in a surgically induced sepsis model in rats., Materials and Methods: Twenty-two adult male Sprague Dawley rats were included in the study. The cecal ligation and puncture procedure was performed to induce sepsis model. Apomorphine-induced stereotypy test was achieved 24 h after cecal ligation and puncture surgery and then, blood and brain samples were collected for biochemical measurements., Results: Significantly higher stereotypy score was found in sepsis group than in the sham group (P = 0.008). Furthermore, septic rats revealed significantly higher plasma TNF-α (P = 0.002) and malondialdehyde levels (P = 0.002), and brain homovanillic acid (P = 0.004) compared with sham rats. There was a significant and positive correlation between the behavioral and biochemical parameters., Conclusions: Taken together, these results demonstrate the association between inflammatory response, oxidative stress, and stereotypic behavior in an experimental sepsis model. More comprehensive experimental and clinical studies are required to clarify the specific mechanisms underlying SAE., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Different grades of genuine and counterfeit Fraxinus excelsior exudates, marketed as natural sweeteners or mild laxatives, were evaluated for their proximate composition and for saccharidic, organic acids, lipidic and phenolic profile by means of GC-MS and (1)H NMR. Genuine samples contained mannitol (39-48 g/100 g, according to the grade), fructose (9-16 g/100 g), glucose (2-3.7 g/100 g), sorbitol (0,5-0,6 g/100 g), galactose (0.02-0.74 g/100 g), oligosaccharides as mannotriose (13-22 g/100 g) and stachyose (1-11 g/100 g), and traces of myo-inositol, mannose, sucrose. On the contrary, counterfeit samples contained mostly mannitol and sorbitol, with traces of fructose, glucose and mannose. Differences in ash, total polyphenolic content and fatty acid composition allowed a quick identification of counterfeit products, confirmed by a distinct mono-, oligosaccharidic and phenolic pattern. Elenolic acid (63-1628 mg/kg), tyrosol (15-774 mg/kg), homovanillic acid (2,39-52.8 mg/Kg), dopaol (0.8-63 mg/kg), pinoresinol (4.2-18.5 mg/kg) and fraxetin (0.25-11.64 mg/kg), albeit showing a wide concentration range, were the most abundant substances detected in the phenolic fraction of Fraxinus manna, while esculetin, p-hydroxybenzoic acid, 4-hydroxyphenacetic acid, 3,4 hydroxybenzoic acid, hydroxy-pinoresinol, medioresinol and siringaresinol were present in low amounts. The polyphenolic profile may be used as a marker for authentication and should be considered in the evaluation of nutritional and health properties ascribed to Fraxinus manna., (© 2013.)

The prevalence of childhood obesity has risen dramatically and coincident with this upsurge is a growth in adverse childhood psychological conditions including impulsivity, depression, anxiety and attention deficit/hyperactive disorder (ADHD). Due to confounds that exist when determining causality of childhood behavioral perturbations, controversy remains as to whether overnutrition and/or childhood obesity is important. Therefore, we examined juvenile mice to determine if biobehaviors were impacted by a short-term feeding (1-3wks) of a high-fat diet (HFD). After 1wk of a HFD feeding, mouse burrowing and spontaneous wheel running were increased while mouse exploration of the open quadrants of a zero maze, perfect alternations in a Y-maze and recognition of a novel object were impaired. Examination of mouse cortex, hippocampus and hypothalamus for dopamine and its metabolites demonstrated increased homovanillic acid (HVA) concentrations in the hippocampus and cortex that were associated with decreased cortical BDNF gene expression. In contrast, pro-inflammatory cytokine gene transcripts and serum IL-1α, IL-1β, TNF-α and IL-6 were unaffected by the short-term HFD feeding. Administration to mice of the psychostimulant methylphenidate prevented HFD-dependent impairment of learning/memory. HFD learning/memory impairment was not inhibited by the anti-depressants desipramine or reboxetine nor was it blocked in IDO or IL-1R1 knockout mice. In sum, a HFD rapidly impacts dopamine metabolism in the brain appearing to trigger anxiety-like behaviors and learning/memory impairments prior to the onset of weight gain and/or pre-diabetes. Thus, overnutrition due to fats may be central to childhood psychological perturbations such as anxiety and ADHD., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The applicability of microbore ultrahigh performance liquid chromatography (UHPLC) with electrochemical detection for offline analysis of a number of well-known neurotransmitters in less than 10 μL microdialysis fractions is described. Two methods are presented for the analysis of monoamine or amino acid neurotransmitters, using the same UHPLC instrument. Speed of analysis of noradrenaline (NA), dopamine (DA), serotonin (5-HT), and the metabolites homovanillic acid (HVA), 5-hydroxyindole aceticacid (5-HIAA), and 3,4-dihydroxyphenylacetic acid (DOPAC) was predominated by the retention behavior of NA, the nonideal behavior of matrix components, and the loss in signal of 5-HT. This method was optimized to meet the requirements for detection sensitivity and minimizing the size of collected fractions, which determines temporal resolution in microdialysis. The amino acid neurotransmitters glutamate (Glu) and γ-aminobutyric acid (GABA) were analyzed after an automated derivatization procedure. Under optimized conditions, Glu was resolved from a number of early eluting system peaks, while the total runtime was decreased to 15 min by a 4-fold increase of the flow rate under UHPLC conditions. The detection limit for Glu and GABA was 10 nmol/L (15 fmol in 1.5 μL); the monoamine neurotransmitters had a detection limit between 32 and 83 pmol/L (0.16-0.42 fmol in 5 μL) in standard solutions. Using UHPLC, the analysis times varied from 15 min to less than 2 min depending on the complexity of the samples and the substances to be analyzed.

Experimental and clinical data suggest that the Ginkgo biloba standardized extract EGb 761® exerts beneficial effects in conditions which are associated with impaired cognitive function. However, the neurochemical correlates of these memory enhancing effects are not yet fully clarified. The aim of this study was to examine the effect of repeated oral administration of EGb 761® and some of its characteristic constituents on extracellular levels of dopamine (DA), noradrenaline (NA), serotonin (5-HT), acetylcholine (ACh) and the metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in the medial prefrontal cortex (mPFC) of awake rats by use of in vivo microdialysis technique. Subacute (14 days, once daily), but not acute, oral treatment with EGb 761® (100 and 300 mg/kg) or the flavonoid fraction, which represents about 24% of the whole extract caused a significant and dose-dependent increase in extracellular DA levels in the mPFC. Repeated administration of EGb 761® also caused a modest but significant increase in the NA levels, whereas the concentrations of 5-HT and those of the metabolites DOPAC, HVA and 5-HIAA were not affected. The same treatment regimen was used in a subsequent study with the aim of investigating the effects of two Ginkgo-specific acylated flavonols, 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-β-D-glucosyl)-α-L-rhamnosyl)quercetin (Q-ag) and 3-O-(2''-O-(6'''-O-(p-hydroxy-trans-cinnamoyl)-β-D-glucosyl)-α-L-rhamnosyl)kaempferol (K-ag). Both compounds together represent about 4.5% of the whole extract. Repeated oral treatment with Q-ag (10 mg/kg) for 14 days caused a significant increase in extracellular DA levels of 159% and extracellular acetylcholine (ACh) levels of 151% compared to controls. Similarly, administration of K-ag (10 mg/kg) induced a significant rise of DA levels to 142% and ACh levels to 165% of controls, whereas treatment with isorhamnetin, an O-methylated aglycon component of EGb 761® flavonol glycosides had no effect. None of the tested flavonoids had a significant effect on extracellular DOPAC and HVA levels. The present findings provide evidence that the subacute treatment with EGb 761® and its flavonol constituents increases DA and ACh release in the rat mPFC, and suggest that the two Ginkgo-specific acylated flavonol glycosides Q-ag and K-ag are active constituents contributing to these effects. As seen for isorhamnetin, the effect on neurotransmitter levels seems not to be a general effect of flavonols but rather to be a specific action of acylated flavonol glycosides which are present in EGb 761®. The direct involvement of these two flavonol derivatives in the increase of dopaminergic and cholinergic neurotransmission in the prefrontal cortex may be one of the underlying mechanisms behind the reported effects of EGb 761® on the improvement of cognitive function.

Objective: Ningdong granule is a traditional Chinese medicine preparation for the treatment of Tourette's syndrome., Methods: Sixty-four rats were randomly assigned to a control group and three experimental groups, respectively. Rat models of Tourette's syndrome were established via intraperitoneal injection of apomorphine (Apo). The rats in the experimental groups were subsequently intragastrically injected with haloperidol at 10 mg/kg (haloperidol group), ningdong granule at 370 mg/kg (NDG group), and normal saline (0.9%) at 10 mL/kg (Apo group), respectively. Rat behaviors were observed and recorded on a daily basis. After 12 w, all rats were sacrificed, and sera and striatal tissues were harvested. Homovanillic acid levels in sera, as well as dopamine and dopamine D2 receptor mRNA expression in the striatum, were measured to determine possible mechanisms of Ningdong granule on the dopamine system in a rat model ofTourette's syndrome., Results: Following intervention, stereotype actions of the Tourette's syndrome rats were significantly inhibited in the haloperidol and NDG groups, respectively (P < 0.01). Homovanillic levels were significantly greater in the haloperidol and NDG groups, respectively (P < 0.05). In addition, dopamine levels were significantly less in the NDG group (P < 0.01), and DRD2 mRNA expression was significantly reduced in the haloperidol and NDG groups, respectively (P < 0.05)., Conclusion: Results demonstrated that Ning-dong granule effectively inhibited stereotype actions and Tourette's syndrome symptoms by promoting dopamine metabolism, reducing dopamine levels in the striatum, increasing homovanillic acid content in sera, and reducing mRNA expression of DRD2 in the striatum.

Major depressive disorder is a prevalent disease, and current pharmacotherapy is considered to be inadequate. It has been hypothesized that a triple reuptake inhibitor (TRI) that activates dopamine (DA) neurotransmission in addition to serotonin and norepinephrine (NE) circuitries may result in enhanced antidepressant effects. Here, we investigated the pharmacological effects of a serotonin-preferring TRI-amitifadine (EB-1010, formerly DOV 21947). The effects of amitifadine (10 mg/kg ip.) on extracellular concentrations of monoamines and their metabolites in rat brain regions were investigated using the in vivo microdialysis technique. The effects of amitifadine on locomotor activity and stereotyped behavior were also evaluated. A major metabolite of amitifadine, the 2-lactam compound, was investigated for inhibition of monoamine uptake processes. Amitifadine markedly and persistently increased extracellular concentrations of serotonin, NE, and DA in prefrontal cortex. The extracellular concentrations of DA were also increased in the DA-rich areas striatum and nucleus accumbens. The extracellular concentrations of the metabolites of serotonin, 5-hydroxyindoleacetic acid, and DA, 3,4-dihydroxyphenylacetic and homovanillic acid, were also markedly decreased in brain regions. Amitifadine did not increase locomotor activity or stereotypical behaviors over a broad dose range. The lactam metabolite of amitifadine weakly inhibited monoamine uptake. Thus, amitifadine increased extracellular concentrations of serotonin, NE, and DA, consistent with TRI. Although amitifadine significantly increased DA in the nucleus accumbens, it did not induce locomotor hyperactivity or stereotypical behaviors. The enhancement of serotonin, NE, and DA in rat brain regions associated with depression suggest that amitifadine may have novel antidepressant activity., (Copyright © 2011 Wiley Periodicals, Inc.)

An extensive literature has documented adverse effects on mental health in anabolic androgenic steroids (AAS) abusers. Depression seems a common adverse reaction in AAS abusers. Recently it has been reported that in a rat model of AAS abuse stanozolol induces behavioural and biochemical changes related to the pathophysiology of major depressive disorder. In the present study, we used the model of AAS abuse to examine possible changes in the monoaminergic system, a neurobiological substrate of depression, in different brain areas of stanozolol-treated animals. Wistar rats received repeated injections of stanozolol (5mg/kg, s.c.), or vehicle (propylene glycol, 1ml/kg) once daily for 4weeks. Twenty-four hours after last injection, changes of dopamine (DA) and relative metabolite levels, homovanilic acid (HVA) and 3,4-dihydroxy phenylacetic acid (DOPAC), serotonin (5-HT) and its metabolite levels, 5-hydroxy indolacetic acid (5-HIAA), and noradrenaline (NA) amount were investigated in prefrontal cortex (PFC), nucleus accumbens (NAC), striatum (STR) and hippocampus (HIPP). The analysis of data showed that after chronic stanozolol, DA levels were increased in the HIPP and decreased in the PFC. No significant changes were observed in the STR or in the NAC. 5-HT and 5-HIAA levels were decreased in all brain areas investigated after stanozolol exposure; however, the 5-HIAA/5-HT ratio was not altered. Taken together, our data indicate that chronic use of stanozolol significantly affects brain monoamines leading to neurochemical modifications possibly involved in depression and stress-related states., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In a previous study, we confirmed that orally administered L-ornithine can be transported into the brain of mice. In addition, orally administered L-ornithine, within a limited dose range, had an anxiolytic-like effect in the elevated plus-maze test. However, the mechanism by which orally administered L-ornithine reduced the stress response in mice is still unclear. Experiment 1 determined whether orally administered L-ornithine could reduce the stress-induced activation of hypothalamic-pituitary-adrenal axis. Mice were orally administered L-ornithine (0, 0.75, 1.5 and 3 mmol/10 ml/kg, p.o.), and restrained for 30 min from 30 min post administration. There was a significant decrease in the corticosterone levels in the group receiving 0.75 mmol of L-ornithine compared to the control group. In Experiment 2, the effect of orally administered L-ornithine (0 and 0.75 mmol/10 ml/kg, p.o.) on endogenous monoamine release was investigated using in vivo microdialysis. Only the monoamines metabolites 5-hydroxyindoleacetic acid (5-HIAA), dihydroxyphenylacetic acid (DOPAC) and homovallinic acids (HVA) were detected in the present study. Dialysate concentrations of 5-HIAA, DOPAC and HVA were not significantly changed immediately after administration of L-ornithine and restraint stress. In conclusion, changes of corticosterone concentrations by orally administered L-ornithine were not related to alterations in brain monoamine metabolisms., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Electrochemical detection is often used to detect catecholamines and indolamines in brain samples that have been separated by conventional reverse-phase high performance liquid chromatography (HPLC). This paper presents the transfer of an existing chromatographic method for the determination of monoamines in brain tissues using 5 μm granulometry HPLC columns to columns with a particle diameter less than 3 μm. Several parameters (repeatability, linearity, accuracy, limit of detection, and stability of samples) for this new ultrafast high performance liquid chromatography (UHPLC) method were examined after optimization of the analytical conditions. The separation of seven compounds, noradrenaline, dopamine and three of its metabolites, dihydroxyphenylacetic acid, homovanillic acid, and 3-methoxytyramine, and serotonin and its metabolite, 5-hydroxyindole-3-acetic acid was analyzed using this UHPLC-electrochemical detection method. The final method, which was applied to brain tissue extracts from mice, rats, and cats, decreased analysis time by a factor of 4 compared to HPLC, while guaranteeing good analytical performance., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Our aim was to assess biochemical parameters to detect choroid plexus dysfunction in Kearns-Sayre syndrome (KSS) patients. We studied CSF from 7 patients with KSS including total proteins, 5-methyltetrahydrofolate, homovanillic acid (HVA) and Selenium (Se) concentrations. High Se values, increased HVA and total protein concentrations and decreased 5-MTHF values were observed in all cases. This pattern seems very specific to KSS since it was only detected in 7 patients out of 1850 CSF samples analysed, and may represent a good biochemical model for evaluating choroid plexus dysfunction. The accumulated Se in CSF might have deleterious consequences such as toxicity effects., (Copyright © 2011 © Elsevier B.V. and Mitochondria Research Society. All rights reserved. Published by Elsevier B.V. All rights reserved.)

A sensitive assay method was developed for a parallel, rapid and precise determination of dopamine and its metabolites, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid, from brain microdialysates. The method consisted of a pre-treatment step, freeze-drying (lyophilization), to concentrate dopamine and its metabolites from the microdialysates, and a detection step using liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). In particular, the reaction monitoring mode was selected for its extremely high degree of selectivity and the stable-isotope-dilution assay for its high precision of quantification. The developed method was characterized by the following parameters: the precision of the developed method was determined as ≥88.6% for dopamine, ≥89.9% for homovanillic acid, ≥86.1% for 3-methoxytyramine and ≥88.1% for 3,4-dihydroxyphenylacetic acid; the mean accuracy was determined as ≥88.2% for dopamine, ≥88.3% for homovanillic acid, ≥85.9% for 3-methoxytyramine and ≥88.6% for 3,4-dihydroxyphenylacetic acid. The developed method was compared to (1) other combinations of pre-treatment methods (solid phase extraction and nitrogen stripping) with LC-MS and (2) another detection method, liquid chromatography, with electrochemical detection. The novel developed method using combination of lyophilization with LC-ESI-MS/MS was tested on real samples obtained from the nucleus accumbens of rat pups after an acute methamphetamine administration. It was proven that the developed assay could be applied to both a simultaneous analysis of all four substrates (dopamine, homovanillic acid, 3-methoxytyramine and 3,4-dihydroxyphenylacetic acid) in microdialysis samples acquired from the rat brain and the monitoring of their slight concentration changes on a picogram level over time following methamphetamine stimulus., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We wished to determine whether L-DOPA, a common treatment for the motor deficits in Parkinson's disease, could also reverse the motor deficits that occur during aging. We assessed motor performance in young (2-3 months) and old (20-21 months) male C57BL/6 mice using the challenge beam and cylinder tests. Prior to testing, mice were treated with L-DOPA or vehicle. Following testing, striatal tissue was analyzed for phenotypic markers of dopamine neurons: dopamine, dopamine transporter, and tyrosine hydroxylase. Although the dopaminergic markers were unchanged with age or L-DOPA treatment, L-DOPA reversed the motor deficits in the old animals such that their motor coordination was that of a young mice. These findings suggest that some of the locomotor deficits that accompany normal aging are responsive to L-DOPA treatment and may be due to subtle alterations in dopaminergic signaling., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

In this paper a structure of a microdialytic cannula inserted into brain areas just before a microdialysis is described. The cannula used allowed to find out a correspondence of behavioral and biochemical changes in C57BL/6 mice at various time intervals after a single dose administration (20 mg/kg) of the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, without any additional pharmacological actions enhancing an extracellular striatal dopamine concentration. Immediately after 1-methyl-4-phenyl-1.2,3.6-tetrahydropyridine administration an essential disturbance of mice behavior and a significant reduction of the extracellular concentration of dopamine and homovanillic acid were observed in striatum. A week after the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration neither behavior nor the extracellular dopamine and homovanillic acid striatal concentration substantially differed from those of controls. 30 days after the neurotoxin administration there was again an essential disturbance of behavior and the large reduction of dopamine and its metabolite concentration in striatum. There was suggested that a reduction of the dopamine concentration immediately after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injection connected with abnormalities of dopamine synthesis and metabolism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine whereas a reduction of the extracellular striatal dopamine concentration 30 days after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine administration related to damage of the nigrastriatal dopaminergic system.

The effects of different types of pesticides on the in vivo striatal dopamine release were investigated by using in vivo brain microdialysis technique. MPTP, paraquat, maneb, dicofol, DDT, lindane and flutriafol (1 mM) were administered directly into the striatum, and levels of dopamine and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovallinic acid (HVA) were measured using HPLC-EC. Intrastriatal administration of pesticides induced the following maximal effects on the dopamine levels: maneb 791 +/- 87%, dicofol 101 +/- 1%, DDT 779 +/- 32%, paraquat 956 +/- 80%, lindane 281 +/- 28% and flutriafol 218 +/- 51% with respect to basal levels. Infusion of pesticides also produced alterations in extracellular DOPAC and HVA levels. A comparative scale of potency was developed to estimate the relative potency of pesticides to induce striatal dopamine release in vivo, using the same concentration and experimental conditions. According to this comparative scale of potency, paraquat is 10 times more potent (in a scale of 10) than dicofol, which did not induce any effect on dopamine release. The second more potent pesticide is maneb, followed by DDT, the organochlorine which has the highest potential to produce alterations on dopaminergic neurotransmission; flutriafol and the organochlorine lindane produced moderate increases in dopamine levels. These results suggest that different classes of pesticides, with different structures and biochemical activities, may affect the striatal dopaminergic system differently, inducing neurotoxicity.

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) were selectively determined by quartz crystal nanobalance sensor in conjunction with net analyte signal (NAS)-based method called HLA/GO. An orthogonal design was applied for the formation of calibration and prediction sets including HVA, VMA, and some common and structurally similar urine compounds. The selection of the optimal time range involved the calculation of the NAS regression plot in any considered time window for each test sample. The searching of a region with maximum linearity of NAS regression plot (minimum error indicator) and minimum of predicted error sum of squares value was carried out by applying a moving window strategy. Based on the obtained results, the differences on the adsorption profiles in the time range between 1 and 300 s were used to determine mixtures of compounds by HLA/GO method. Several figures of merit like selectivity, sensitivity, analytical sensitivity, and limit of detection were calculated for both compounds. The results showed that the method was successfully applied for the determination of VMA and HVA.

Biogenic amines (norepinephrine, dopamine, homovanillic acid, serotonin and 5-hyroxyindole acetic acid) were measured by HPLC method in adult F1 generation rats' brain regions (brainstem, hypothalamus, hippocampus, striatum and frontal cortex), whose mothers (P generation) were treated with vitamin A or vitamin D neonatally (hormonal imprinting). Many significant differences were found, related to the maternally untreated controls. In the earlier studied P generation females, vitamin A consistently influenced the serotonerg system (5HIAA), while vitamin D the dopaminerg system (DA or HVA). Vitamin A imprinting always resulted in reduced, while that by vitamin D always in increased tissue levels. In the present case (directly untreated F1 generation) the transgenerational effect was not unidirectional, however biogenic amine tissue levels were strongly disturbed and brain-area dependent. The results call attention to the transgenerational effect of hormonal imprinting in the case of receptor level acting vitamins which are frequently used in the most imprinting-sensitive period (perinatally) of human life and suggests that caution is warranted.

Ethnopharmacological Relevance: Withania somnifera root extract (Ws)/Ashwagandha/Indian ginseng is a traditional herbal medicine, used over 4000 years in India, shown to have effect on neural growth and locomotor function. Although catecholamines and oxidative stress resulting in neurodegeneration and locomotor disorder are the main events in Parkinson's disease (PD), efficacy of the drug on these molecules and physiological abnormality are not clear., Aim of the Study: The objective of the study was to examine effect of Ws on catecholamines and physiological abnormalities seen in PD using PD model mouse., Materials and Methods: Mouse were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 4 days to show biochemical and physiological abnormalities similar to patients with PD. PD mice were treated with Ws 100mg/kg body weight for 7 or 28 days. Catecholamines: dopamine (DA), 3,4-dihydroxy-phenylacetic acid (DOPAC) and homovanillic acid (HVA); antioxidants: glutathione (GSH) and glutathione peroxidase (GPx); and lipid peroxidation marker (TBARS) were analyzed in the Ws treated and untreated PD mouse striatum., Results: Mouse treated with MPTP showed reduced levels of DA, DOPAC, HVA, GSH and GPx and induced thiobarbituric acid reactive substance (TBARS) level compared to the control. Physiological abnormalities were seen in the mouse as determined by hang test and rotarod test. Oral treatment of PD mouse Ws root extract (100mg/kg body weight) for 7 days or 28 days increased DA, DOPAC and HVA levels and normalized TBARS levels in the corpus striatum of the PD mouse. The 7 days Ws treated mice showed improved motor function as determined by hang test and rotarod test. Treatment with Ws for 28 days increased GSH and GPx levels in the striatum compared to the Ws untreated PD mouse striatum., Conclusion: These data suggest that Ws is a potential drug in treating catecholamines, oxidative damage and physiological abnormalities seen in the PD mouse.

The high-fat ketogenic diet (KD) leads to an increase of blood ketone bodies (KB) level and has been used to treat refractory childhood seizures for over 80 years. Recent reports show that KD, KB and their components (d-beta-hydroxybutyrate, acetoacetate and acetone) have neuroprotective for acute and chronic neurological disorders. In our present work, we examined whether KD protected dopaminergic neurons of substantia nigra (SN) against 6-hydroxydopamine (6-OHDA) neurotoxicity in a rat model of Parkinson's disease (PD) using Nissl staining and tyrosine hydroxylase (TH) immunohistochemistry. At the same time we measured dopamine (DA) and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum. To elucidate the mechanism, we also measured the level of glutathione (GSH) of striatum. Our data showed that Nissl and TH-positive neurons increased in rats fed with KD compared to rats with normal diet (ND) after intrastriatal 6-OHDA injection, so did DA and its metabolite DOPAC. While HVA had not changed significantly. The change of GSH was significantly similar to DA. We concluded that KD had neuroprotective against 6-OHDA neurotoxicity and in this period GSH played an important role.

Evidence for involvement of cytochrome P450 2E1 in the MPTP-induced mouse model of PD has been reported [Vaglini, F., Pardini, C., Viaggi, C., Bartoli, C., Dinucci, D., Corsini, G.U., 2004. Involvement of cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease. J. Neurochem. 91, 285-298]. We studied the sensitivity of Cyp2e1(-/-) mice to the acute administration of MPTP in comparison with their wild-type counterparts. In Cyp2e1(-/-) mice, the reduction of striatal DA content was less pronounced 7 days after MPTP treatment compared to treated wild-type mice. Similarly, TH immunoreactivity analysis of the substantia nigra of Cyp2e1(-/-) mice did not show any neuronal lesions after MPTP treatment. In contrast to this, wild-type animals showed a minimal but significant lesioning by the toxin as evaluated also by means of non-stereologic computerized assisted analysis of this brain area. Striatal levels of DA metabolites after 7 days were variably affected by the toxin, but consistent differences between the two animal strains were not observed. We evaluated short-term changes in the levels of striatal DA and its metabolites, and we monitored striatal MPP(+) levels. Striatal MPP(+) was cleared more rapidly in Cyp2e1(-/-) mice than in wild-type animals and, consistently, striatal DA content decreased faster in Cyp2e1(-/-) mice than in wild-type animals, and 3-methoxytyramine and HVA levels showed an early and sharp rise. Our findings suggest that Cyp2e1(-/-) mice are weakly sensitive to MPTP-induced brain lesions, markedly in contrast with a protective role of the enzyme as suggested previously. The differences observed between the knockout mice and their wild-type counterparts are modest and may be due to an efficient compensatory mechanism or genetic drift in the colonies.

The aim of the present study was to investigate whether high fat consumption changes the effects of stress on both motor activity performance, striatal and cortical dopamine and serotonin metabolites in rats. The animals were fed either with high fat or standard diet for 4 weeks. Restraint stress lasting for 15 min at +4 degrees C was applied daily to stress-exposed groups. Motor activity performance was measured weekly by using motor activity monitoring systems. At the end of the study, homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) levels of the striatum and cerebral cortex were measured by HPLCEC. It was observed that restraint stress increased locomotor activity and high-fat diet prevented this effect. Stress and high-fat intake had an additive decreasing effect on striatal HVA levels. 5-HIAA levels, on the other hand, were lower in both high fat and high fat + stress groups compared to the stress group. These results suggest that high-fat intake differentially affected the stress response on striatal dopaminergic and serotonergic neurons in rat brain regions studied and this may be related to the effects observed in motor activity performance.

The purpose of this study was to clarify the influence of different vibration frequencies on wakefulness level. Subjects were 7 healthy male university students aged 21.9+/-1.6 years (mean). All students were non-smokers. Three exposure conditions were used (10 Hz vibration, 20 Hz vibration, and no vibration). Whole-body vertical vibration was applied to subjects sitting on a car passenger seat using a whole-body vibration shaker (CV-300, Akashi) at a single frequency (10 or 20 Hz) at an acceleration level of 0.3 ms(-2) r.m.s. for 24 min. The objective wakefulness level based on EEGs was evaluated in terms of the alpha attenuation coefficient (AAC) obtained by the Alpha Attenuation Test (AAT). As parameters of psychological stress, salivary 3-methoxy-4-hydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were used. The subjective wakefulness level was evaluated using a questionnaire based on the Kwansei Gakuin Sleepiness Scale (KSS), which is a scale developed for the Japanese based on the Stanford Sleepiness Scale (SSS). The KSS score, representing the subjective wakefulness level, decreased after the exposure irrespective of the exposure condition, but the decrease was not significant. The AAC, representing the objective wakefulness level, significantly decreased only after vibration exposure (10 Hz/20 Hz) but did not differ between the two vibration frequencies. No significant changes were observed after exposure to whole-body vibration in MHPG or HVA as parameters of vibration-related stress. The AAC decreased after exposure to whole-body vibration (10 Hz/20 Hz), suggesting a decrease in the wakefulness level. However, no differences were observed in the influence of the two different vibration frequencies test.

Brain-derived neurotrophic factor (BDNF) has been shown to influence monoamine transmitter synthesis, metabolism and release. We investigated possible relationships between four BDNF gene polymorphisms and cerebrospinal fluid (CSF) concentrations of 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), and 3-methoxy-4-hydroxyphenylglycol (MHPG) in healthy volunteers (n = 132). All BDNF polymorphisms (270 C/T, -633 T/A, Val66Met, and 11757 G/C) were associated with MHPG (P < 0.02), but not with 5-HIAA and HVA concentrations. At a second clinical investigation 8-20 years after CSF sampling 30% of the subjects had experienced various psychiatric disorders. Development of a psychiatric disorder was predicted by low 5-HIAA concentrations (P = 0.01). The results suggest that BDNF gene variation participates in regulation of norepinephrine turnover rates in the central nervous system of human subjects.

Objectives: The present study investigates the effects of injections of a specific N-methyl-D-aspartic acid (NMDA) antagonist 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phophonic acid (CPP) into the prefrontal cortex (PFC) on the extracellular concentrations of dopamine and acetylcholine in the nucleus accumbens (NAc) and on motor activity in the freely moving rat., Materials and Methods: Sprague-Dawley male rats were implanted with guide cannulas into the medial PFC and NAc to perform bilateral microinjections and microdialysis experiments. Spontaneous motor activity was monitored in the open field., Results: Injections of CPP (1 microg/0.5 microL) into the PFC produced a significant increase of the baseline extracellular concentrations of dopamine (up to 130%), dihydroxyphenylacetic acid (DOPAC; up to 120%), homovanillic acid (HVA; up to 130%), and acetylcholine (up to 190%) in the NAc as well as motor hyperactivity. In the NAc, perfusion of the NMDA and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate antagonists CPP (50 microM)+6,7-dinitroquinoxaline-2,3-dione (DNQX; 50 microM) through the microdialysis probe blocked acetylcholine release, but not DOPAC and HVA increases produced by CPP injections into the PFC. Also, increases in motor activity produced by prefrontal injections of CPP were significantly reduced by bilateral injections into the NAc of a mixed D1/D2 antagonist, flupenthixol (5 and 25 microg/0.5 microL). Injections into the NAc of the muscarinic antagonist scopolamine (1 and 10 microg/0.5 microL) further increased, and of the nicotinic antagonist mecamylamine (1 and 10 microg/0.5 microL) did not change, the increases in motor activity produced by prefrontal CPP injections., Conclusions: These results suggest that the dysfunction of NMDA receptors in the PFC could be a key factor in the neurochemical and motor effects associated with corticolimbic hyperactivity.

This manuscript describes dynamic coating of capillaries with poly(L-lysine) (PLL) and silica nanoparticles (SiO2 NPs) and use of the as-prepared capillaries for the separation of biogenic amines and acids by CE in conjunction with LIF detection. The directions of EOF are controlled by varying the outmost layer of the capillaries with PLL and SiO2 NPs, respectively. Over the pH range 3.0-5.0, the (PLL-SiO2NP)n-PLL capillaries have an EOF toward the anodic end and are more suitable for the separation of acids with respect to speed, while the (PLL-SiO2NP)n capillaries have an EOF toward the cathodic end and are more suitable for the separation of biogenic amines regarding speed and sensitivity. The separations of standard solutions containing five amines and two acids by CE with LIF detection using (PLL-SiO2NP)2-PLL and (PLL-SiO2NP)3 capillaries were accomplished within 10 and 7 min, providing plate numbers of 3.8 and 5.0x10(4) plates/m for 5-hydroxytryptamine (5-HT), respectively. The LODs for 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) are 32 and 2 nM and 0.2 and 1.5 nM when using the (PLL-SiO2NP)2-PLL and (PLL-SiO2NP)3 capillaries, respectively. Identification and quantification of 5-HIAA, homovanillic acid, and DL-vanillomandelic acid in urine samples from a male before and after drinking green tea were tested to validate practicality of the present approach. The results show that the (PLL-SiO2NP)2-PLL capillary provides greater resolving power, while the (PLL-SiO2NP)3 capillary provides better sensitivity, higher efficiency, and longer durability for the separation of the amines and acids.

Rationale: Tardive dyskinesia is a syndrome of abnormal and involuntary movements which occurs as a complication of long-term neuroleptic therapy especially classical neuroleptics such as haloperidol and chlorpromazine. Dysfunction of GABA receptor mediated inhibition, and increased glutamatergic neurotransmission has been implicated in the development of orofacial dyskinesia in rats and tardive dyskinesia in humans. Neurosteroids modulate both GABAergic as well as glutamatergic neurotransmission in various brain areas., Objective: The objective of the present study was to elucidate the role of various neurosteroids in neuroleptic-induced vacuous chewing movements and related behaviors in rats by using behavioral, biochemical, and neurochemical parameters., Materials and Methods: Animals chronically treated with haloperidol (1 mg/kg i.p.) for a period of 21 days exhibited marked increase in vacuous chewing movements, tongue protrusions, and facial jerkings as compared to vehicle-treated controls. It also resulted in increased superoxide anion levels and lipid peroxidation, whereas decreased levels of endogenous antioxidant enzymes (catalase and superoxide dismutase) in rat brain striatum homogenates. Neurochemical studies revealed that chronic administration of haloperidol resulted in significant decrease in the levels of dopamine, serotonin, and norepinephrine in rat brain striatum homogenates, whereas urine biogenic amines metabolite levels were increased. In a series of experiments, rats co-administered with allopregnanolone (0.5, 1, and 2 mg/kg i.p.) and progesterone (5, 10, and 20 mg/kg i.p.), both positive GABA-modulating [negative N-methyl-D-aspartate (NMDA)-modulating] neurosteroids prevented, whereas pregnenolone (0.5, 1, and 2 mg/kg i.p.) and dihydroxyepiandrosterone sulfate (0.5, 1, and 2 mg/kg i.p.) both negative GABA-modulating (positive NMDA-modulating) neurosteroids aggravated all the behavioral, biochemical, and neurochemical parameters., Conclusions: These results suggest that neurosteroids may play a significant role in the pathophysiology of vacuous chewing movements and related behaviors by virtue of their action on either the GABA or NMDA modulation. Furthermore, neurosteroids showing selectivity for positive GABA modulation and/or negative NMDA modulation may be particularly efficacious as novel therapeutic agents for the treatment of tardive dyskinesia and deserve further evaluation.

Rationale and Objectives: Flinders sensitive line (FSL) rats, an animal model of depression, display a different pattern of maternal behavior compared to Sprague-Dawley (SD) controls. In this study, we examined the rewarding value of mother-infant interaction for FSL dams., Materials and Methods: In the main study, we measured monoamine levels in the nucleus accumbens (NAc) of early postpartum FSL and SD dams during an interaction with pups, using the microdialysis technique. In addition, we compared the preference patterns of FSL and SD rats using the conditioned place preference paradigm, with pups as the unconditioned stimuli., Results: Dopamine (DA) levels in dialysates from the NAc of SD dams but not FSL dams were elevated while interacting with pups but the metabolism of DA to dihydroxyphenylacetic acid was greater in FSL than in SD dams. While SD dams showed a conditioned preference for a region that was associated with SD pups, FSL dams did not show a preference for regions associated either with SD or FSL pups, but water deprived FSL rats demonstrated a preference to a region associated with water, eliminating an alternative explanation of learning deficit in FSL rats., Conclusions: Taken together, these results suggest that FSL dams are less rewarded by pups, compared to control dams.

We investigated the effects of echinacoside, a phenylethanoid glycoside isolated and purified from the stems of Cistanche salsa, a Chinese herbal medicine, on the striatal extracellular levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in 6-hydroxydopamine (6-OHDA) lesion rats. Seven days after 6-OHDA was injected into the right striatum of rats, the striatal extracellular levels of DA, DOPAC and HVA fell significantly (P<0.01 vs. vehicle), as demonstrated by the method of cerebral microdialysis and high performance liquid chromatography with electrochemical detection. However, simultaneous treatment with echinacoside (7.0, 3.5mg/kg) attenuated the diminution of them (P<0.01 vs. model). The results implied that echinacoside could protect the striatal dopaminergic neurons from injury induced by 6-OHDA and may be useful in the prevention and treatment of Parkinson's disease (PD).

Lower tissue levels of dopamine and 5-hydroxytryptamine (5-HT) have been found in the nucleus accumbens of alcohol-naïve rats selectively bred to prefer ethanol than in rats bred to avoid it. These findings have led to the hypothesis that differences in the dopamine and 5-HT tone may be linked to ethanol preference. In the present study we used the in vivo microdialysis technique to determine the actual extracellular levels of dopamine, its metabolites 3,4-dihydroxyphenyl acetaldehyde (DOPALD), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-HT and 5-hydroxyindolacetic acid (5-HIAA) in the shell of nucleus accumbens of rat lines selectively bred as either high-ethanol (UChB) or low-ethanol (UChA) drinkers. Basal extracellular levels of dopamine, DOPALD, DOPAC and HVA were lower in the shell of nucleus accumbens of ethanol-naïve UChB than in UChA rats. In agreement, when perfused with 100 microM d-amphetamine or 100 mM KCl lower dopamine increases were observed in nucleus accumbens of UChB rats compared to UChA rats, indicating lower cytosolic (d-amphetamine releasable) and vesicular (KCl releasable) dopamine pools in UChB animals. Since the experiments were performed in ethanol-naïve rats, the present results suggest an innate deficiency in the mesolimbic dopamine system of UChB rats. There were no line differences in basal, d-amphetamine or KCl stimulated 5-HT levels. Thus, the present findings support a role of dopamine, but not of 5-HT, as predictor of ethanol preference in UChB rats. Overall, data obtained are in agreement with previous reports in other rat lines showing that lower dopamine levels and its metabolites are associated with a genetic predisposition to ethanol preference.

Objective: To develop a mouse model to mimic the behavioral and neurochemical changes of Tourette syndrome (TS) by 1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) induction and to investigate the effects of fluoxetine and haloperidol on head twitch response (HTR) induced by DOI., Methods: 1) Preparation of mouse model of TS: Forty mice were randomly divided into experimental and control groups (n=20 each). DOI (1 mg/kg) was administered by peritoneal injection in the experimental group. The control group was injected with normal saline. The levels of dopamine (DA) and homovanillic acid (HVA), the metabolite of DA, in both groups were measured by high performance liquid chromatography and electrochemical detection. 2) Effects of fluoxetine and haloperidol on HTR: Eighty mice were randomly administered with either fluoxetine (2 mg/kg), haloperidol (0.8 mg/kg), fluoxetine + haloperidol or normal saline. DOI (1 mg/kg) was peritoneally injected 20 minutes later (acute trial) or 18-20 hrs after a 21 days injection of fluoxetine or haloperidol (chronic trial). The frequency of DOI induced HTR was observed immediately after DOI injection., Results: The levels of DA and HVA in the experimental group were significantly lower than those in the control group (DA: 45.00 +/-11.24 ng/mg vs 58.16 +/-14.51 ng/mg; HVA:10.54 +/-1.86 ng/mg vs 12.82 +/-2.66 ng/mg). In both acute and chronic trials, the frequency of DOI-induced HTR decreased significantly in mice administered with haloperidol alone or together with fluoxetine (P < 0.05), but it did not change significantly in mice administered with fluoxetine alone compared with the normal saline group., Conclusions: The levels of DA and HVA are reduced in mice with DOI-induced HTR. DOI-induced mouse mode of HTR can mimic the neurochemical and behavioral changes of TS paritially. Haloperidol can inhibit DOI-induced HTR in mice, but fluoxetine can not.

The present study evaluated the effects of cyclic variations of hydrostatic pressure (HP) on neurotransmitters in the whole brain of flounder. The concentrations of the biogenic amines L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine (DA), norepinephrine (NE), epinephrine (E), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxytryptamine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) were measured. Fish were subjected to HP cyclic variations which mimic naturally occurring conditions for a period of 14 days. DA, NE and 5-HT concentrations were significantly smaller by 21, 24 and 36%, respectively, compared to control fish. The concentrations of monoamine metabolites HVA, 3-MT and 5-HIAA were also smaller than those in control fish. These results suggest that central monoaminergic systems were influenced during long exposure to cyclic HP. The decreases of central neurotransmitters content might be involved in the physiological and behavioral responses to intermittent HP in fish.

The feasibility of using an osmotic pump in place of a syringe pump for microdialysis sampling in rat brain was investigated. The use of an osmotic pump permits the rat to be free from the constraints of the standard tethered system. The in vitro flow rates of a microdialysis syringe pump (set at 10.80 microl/h) and the osmotic pump (pump specifications were 11.35 microl/h) with no probe attached were compared, yielding results of 10.87 microl/h+/-1.7% and 10.95 microl/h+/-8.0%, respectively. The average of four flow rate experiments in vivo yielded R.S.D.s less than 10% and an average flow rate of 11.1 microl/h. Following the flow rate studies, in vivo sampling of neurotransmitters was accomplished with the osmotic pump coupled to a microdialysis probe implanted in the brain. Finally, after determination of basal levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the rats, the rats were dosed with benserazide followed by l-3,4-dihydroxyphenylalanine (l-DOPA). The results from the dosing study showed at least a 10-fold increase in compounds in the l-DOPA metabolic pathway (DOPAC and HVA) and a slight or no increase in 5-HIAA (serotonin metabolic pathway.) These results indicate that the osmotic pump is a viable alternative to the syringe pump for use in microdialysis sampling.

We measured the concentrations of monoamines in the brain of Japanese medaka, Oryzias latipes, exposed to tributyltin (TBT). Fish were exposed to 0, 1, 5, 25, or 125 microg g(-1) of TBT via the diet for 21 days. After the administration period, six males and six females in each treatment group were dissected and their brains were collected. The following monoamines were analyzed: dopamine (DA), norepinephrine (NE), epinephrine (E), and 5-hydroxytryptamine (5-HT). The metabolites of DA, 3,4-dihydroxyphenylacetic acid and homovanilic acid, and the metabolite of 5-HT, 5-hydroxyindolacetic acid were also analyzed. The concentration of DA in the brain of male medaka and the concentrations of 5-HT and NE in the female brains were significantly decreased by exposure to 125 microg TBT g(-1). The concentrations of 5-HT and NE in males and of DA in females were slightly decreased by 125 micrg g(-1) of TBT, although the differences were not statistically significant. The present study demonstrates that TBT alters monoamine concentrations in the brain of medaka.

Evidence from several studies suggests that the progressive degeneration of dopaminergic (DA) neurones of the substantia nigra pars compacta (SNc) in Parkinson's disease (PD) may in part be due to excessive release of glutamate from subthalamic projections onto nigral DA neurones. Previous in vitro studies have demonstrated that selective activation of Group III metabotropic glutamate receptors (mGluR) negatively modulates excitatory transmission in the SNc and is neuroprotective against glutamate-mediated toxicity. Consistent with this, we have reported preliminary data indicating that the selective group III mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) can also protect the nigrostriatal system against 6-hydroxydopamine (6-OHDA) toxicity in vivo. We have now extended these preliminary studies in this model and report here that both acute and subchronic intranigral injections of l-AP4 provide significant protection of the nigrostriatal system against 6-OHDA toxicity. This neuroprotection displays a bell-shaped profile with a clear concentration-dependent relationship. In contrast, when administered to animals 7 days post-6-OHDA lesioning, l-AP4 significantly protects the functionality but not the integrity of the nigrostriatal system. We further demonstrate that neuroprotection by l-AP4 in vivo is reversed by coadministration of the selective Group III mGluR antagonist (RS)-alpha-methylserine-O-phosphate, confirming a receptor-mediated mechanism of action. These data provide further compelling evidence that selective activation of Group III mGluR is neuroprotective in an in vivo experimental model of PD, a finding that may have important implications for the future treatment of this disease.

Following our behavioral studies demonstrating augmentation of imipramine action by concomitant administration of nicotine, we investigated the effects of one or 14 days of treatment (twice daily) with imipramine and nicotine on dopamine metabolism in various brain areas of rat and noradrenaline in the brain stem. In addition, we evaluated the responses of this metabolism to apomorphine challenge in the rat. Generally, chronic treatment of imipramine and nicotine produced opposite effects to acute administration. As revealed by HPLC, dopamine metabolism in the nucleus accumbens was slightly decreased after 14 days of treatment with imipramine, and co-administration of nicotine resulted in a significant and much more pronounced depression of dopamine metabolism in all investigated dopaminergic structures. Such biochemical effects suggested the development of a compensatory mechanism related with hypersensitivity of dopamine D(2) receptors in the mesolimbic and nigrostriatal system. Chronic administration of imipramine produced an opposite effect to the acute one in the brain stem noradrenergic system, like it was observed in dopaminergic structures. Significant inhibition of noradrenaline metabolism after acute administration of imipramine may be explained by its inhibitory effect on noradrenaline reuptake process. In contrast, chronic imipramine administration had no effect on noradrenaline metabolism what indicated the development of subsensitivity of (2)-adrenoceptors in the brain stem responsible for the rate of noradrenaline metabolism. Apomorphine alone decreased metabolism of both catecholamine, dopamine and noradrenaline through activation of dopamine D(2) receptors which are located also on noradrenergic neurons. The biochemical response to apomorphine in terms of dopamine metabolism was not changed by chronic administration of the investigated drugs but noradrenaline metabolism in the brain stem was strongly attenuated after a combined treatment of imipramine and nicotine. The present data demonstrate facilitation and potentiation of biochemical antidepressant-like effects of imipramine by nicotine co-treatment. We suggest that nicotine may potentiate the antidepressant-like effects of imipramine by promoting some plastic changes in the brain within dopamine and noradrenaline system considerably more strongly than imipramine alone.

Corticotropin-releasing factor (CRF) plays an important role in stress responses and is mediated through two subtypes of receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2). Each CRF receptor might have a different function through several neurotransmitter systems; however, the mechanism remains unclear. To clarify the role of each receptor in dopamine (DA) metabolism, we measured the change of extracellular concentrations of DA and the metabolites in the ventromedial hypothalamus (VMH) that played important roles in the stress response of freely moving female rats in response to the direct administration of comparative CRFR1 selective agonist, CRF, or CRFR2 selective agonist, Urocortin II (Ucn II), into the brain region. Administration of 10 microg CRF increased extracellular concentrations of DA compared with 2 microg CRF immediately after injection, and this effect was not observed after 60 min of 10 microg CRF injection. On the other hand, this change did not always occur after Ucn II administration. These results suggest that the activation of CRFR1, but not CRFR2, modulates the release of DA in VMH.

Crude extract of kohlrabi (Brassica oleracea gongylodes) was prepared by a simple procedure and its enzymatic activity and total protein concentration were determined. It was found that this crude extract is a rich source of peroxidase (POx) and has high specific activity. Cross-linked polyvinylpyrrolidone was used as a stabilizer in the preparation of the crude extract. The POx activity of kohlrabi crude extract did not vary for at least 2 months when deoxygenated and stored at 4 degrees C. This extract was applied for the spectrofluorometric determination of hydrogen peroxide using homovanillic acid as a fluorogenic substrate. POx catalyzes the hydrogen peroxide oxidation of homovanillic acid to produce a dimer which shows strong fluorescence at 420 nm with excitation at 312 nm. In the optimum conditions, the calibration graph for hydrogen peroxide was linear up to 190 ng mL(-1), with a detection limit of 4.4 ng mL(-1). The relative standard deviation (RSD) was 1.48% for 50 ng mL(-1) hydrogen peroxide. The proposed method was successfully applied to the determination of hydrogen peroxide in honey. The concentration-time profile of H2O2 produced upon dilution of honey was studied and H2O2 contents of some different honeys from various areas of Iran were determined.

Mutations of parkin are linked to early onset Parkinson disease. Here we show that stable transfection of parkin in the human dopaminergic neuroblastoma cell line SH-SY5Y markedly reduced the activities of both monoamine oxidase (MAO) A and B. The amount of 3,4-dihydroxyphenylacetic acid, which is produced during dopamine oxidation by MAO, was greatly reduced by parkin overexpression. Radioligand binding assays showed that MAO binding sites were decreased accordingly. Consistent with these, MAO-B protein level was much lower, whereas the amount of MAO-A protein was not determined due to the lack of a suitable antibody. Co-transfection of either MAO with parkin in HEK293 cells did not significantly alter ubiquitination and degradation of each MAO. When we measured MAO expression by real-time quantitative reverse transcription-PCR, marked reductions were seen in SH-SY5Y cells stably expressing parkin compared with the parental cells or a control line stably transfected with luciferase. In addition, parkin mutants defective in E3 ligase activity exhibited different effects on MAO expression. We found that parkin also significantly decreased mRNA levels of both MAOs in the mouse fibroblast cell line NIH3T3. Furthermore, MAO expression was significantly increased in human B lymphocyte cell lines derived from Parkinson disease patients with homozygous but not heterozygous deletion of exon 4 of parkin. Together these results suggest that parkin suppresses MAO expression. This function may limit the production of reactive oxygen species generated by MAO in dopamine oxidation and would, thus, be beneficial to the survival of dopaminergic neurons.