Low-cost, thin-film, mass-manufacturable carbon electrodes for detection of the neurotransmitter dopamine.

A flexible, thin-film carbon electrode is reported for detection of the key neurotransmitter dopamine using standard electroanalytical techniques of cyclic voltammetry, differential pulse voltammetry and square wave voltammetry. The thin-film electrode has been explored as a possible low-cost solution to detect low concentrations of dopamine and its performance has been compared with a commercially available screen printed carbon electrode. It was found that the thin-film electrode is more sensitive than the screen printed electrode, and can faithfully detect dopamine between 50 pM and 1 mM concentrations. The electrode provides a limit of detection of ~50 pM, displays good selectivity between dopamine and ascorbic acid, and is able to show a level of differentiation between the two compounds in terms of peak currents as well as oxidative potentials at physiologically relevant concentrations. This is in contrast to the screen printed electrode which is unable to discriminate between dopamine and ascorbic acid at the same concentrations. The key advantages of the presented electrode system are its low-cost, flexible substrate, and the ability to achieve very low levels of dopamine detection without requiring any electrode surface modification steps, a key factor in reducing fabrication costs and overall device complexity.

Assessment of gut microbiota fecal metabolites by chromatographic targeted approaches.

Gut microbiota, the specific microbial community of the gastrointestinal tract, by means of the production of microbial metabolites provides the host with several functions affecting metabolic and immunological homeostasis. Insights into the intricate relationships between gut microbiota and the host require not only the understanding of its structure and function but also the measurement of effector molecules acting along the gut microbiota axis. This article reviews the literature on targeted chromatographic approaches in analysis of gut microbiota specific metabolites in feces as the most accessible biological matrix which can directly probe the connection between intestinal bacteria and the (patho)physiology of the holobiont. Together with a discussion on sample collection and preparation, the chromatographic methods targeted to determination of some classes of microbiota-derived metabolites (e.g., short-chain fatty acids, bile acids, low molecular masses amines and polyamines, vitamins, neurotransmitters and related compounds) are discussed and their main characteristics, summarized in Tables.

Diethylation labeling combined with UPLC/MS/MS for simultaneous determination of a panel of monoamine neurotransmitters in rat prefrontal cortex microdialysates.

The primary challenge associated with the development of an LC/MS/MS-based assay for simultaneous determination of biogenic monoamine neurotransmitters such as norepinephrine (NE), dopamine (DA), serotonin (5-HT), and normetanephrine (NM) in rat brain microdialysates is to improve detection sensitivity. In this work, a UPLC/ MS/MS-based method combined with a diethyl labeling technique was developed for simultaneous determination of a panel of monoamines in rat prefrontal cortex microdialysates. The chromatographic run time is 3.5 min/ sample. The limits of detection of the UPLC/MS/MS-based method for NE, DA, 5-HT/ and NM, with/without diethyl labeling of monoamines, are 0.005/0.4 (30/2367 pM), 0.005/0.1 (33/653 pM), 0.005/0.2 (28/1136 pM), and 0.002/0.2 ng/mL (11/1092 pM), respectively. Diethyl labeling of amino groups of monoamines affords 20-100 times increased detection sensitivity of corresponding native monoamines during the UPLC/MS/MS analysis. This could result from the following: (1) improved fragmentation patterns; (2) increased hydrophobicity and concomitantly increased ionization efficiency in ESI MS and MS/MS analysis; (3) reduced matrix interference. This labeling reaction employs a commercially available reagent, acetaldehyde-d4, to label the amine groups on the monoamines via reductive amination. It is also simple, fast (approximately 25-min reaction time), specific, and quantitative under mild reaction conditions. Data are also presented from the application of this assay to monitor the drug-induced changes of monoamine concentrations in rat prefrontal cortex microdialysate samples followed by administration of SKF 81297, a selective D1 dopamine receptor agonist known to elevate the extracellular level of the neurotransmitters DA and NE in the central nervous system.

A dual vulnerability hypothesis for seasonal depression is supported by the seasonal pattern assessment questionnaire in relation to the temperament and character inventory of personality in a general population.

BACKGROUND: Personality structure obtained from the psychobiological Temperament and Character Inventory (TCI) was studied in relation to self-reported seasonal variations in mood and behavior measured by the Seasonal Pattern Assessment Questionnaire (SPAQ). METHODS: The subjects comprised 1761 adults (57.6% women) in the age range 35-85 years, enrolled in the Betula prospective random cohort study of Umea, Sweden. RESULTS: Personality profiles of subjects who reported the occurrence of a high degree of seasonal variation as such were associated with a combination of high self-transcendence (ST) and high persistence (PS), irrespective of the level of harm avoidance (HA). Subjects who reported feeling worst in winter were associated with high HA, irrespective of the levels of ST and PS. Also, subjects feeling worst in summer or experiencing overall problems with seasonal variation were associated with high HA in their personality profiles. Using the SPAQ criteria to define seasonal affective disorder (SAD) or subsyndromal SAD (S-SAD), subjects with these disorders often had combinations of high self-transcendence (ST) and high persistence (PS), but with different associations with HA. LIMITATIONS: No evaluations were made for SAD or subsyndromal SAD according to the DSM-IV or ICD 10 criteria. CONCLUSIONS: Our results relating SPAQ with TCI give support for a dual vulnerability hypothesis for seasonal depression proposed in the literature, where it is attributed to a combination of a seasonal factor and a depression factor. Examining the literature regarding the relationships between the different TCI scales and monoamine neurotransmitter functions, those relationships suggest that these two vulnerability factors for seasonal depression may be modulated by different neurotransmitter systems.

An improved method for analyzing coenzyme Q homologues and multiple detection of rare biological samples.

We have developed a simple method for the estimation of coenzyme Q homologues, neurotransmitters, metal ions, lipid peroxidation, gene expression, and DNA fragmentation simultaneously from genetically engineered mice brain regions and cultured neurons. The primary objective of this study was to improve conventional time-consuming, cumbersome, and less efficient procedures, and reduce the cost of conducting kinetic studies in rare biological samples. The improved method is novel, precise, efficient, accurate, sensitive, economical, versatile, and highly reproducible. The recovery and shelf life of coenzyme Q homologues was significantly increased and the chromatograms exhibited reduced background and retention times. It is envisaged that in addition to coenzyme Q homologues, the improved method could be utilized for the multiple analyses of DNA, RNA and proteins from clinically significant biopsy and autopsy samples.

Quantitative assessment of localization and colocalization of glutamate, aspartate, glycine, and GABA immunoreactivity in the chick retina.

We examined the posthatch chick retina for the frequency of occurrence of localization and colocalization of four amino acid transmitter candidates: glutamate (Glu), aspartate (Asp), gamma aminobutyric acid (GABA), and glycine (Gly) using postembedding methods. We support previous studies of Glu, Asp, GABA, and Gly localization in the direct and indirect functional pathways of the chick retina and extend these studies with new qualitative and quantitative observations. We found that photoreceptors show distinct cellular immunoreactivity for both Glu (Glu+) and Asp+, but not for Gly (Gly-) or GABA. Moreover, there is compartmentalization of Glu and Asp staining within the photoreceptors. All horizontal cells react strongly with Asp and Glu, about three-fourths are GABA+ and three-fourths of these are Gly+. Bipolar cells are uniformly Glu+, heterogeneously Asp+, occasionally Gly+, but GABA-. A majority of amacrine cells stain heterogeneously with all antibodies: 90% are Gly+, slightly more than half colocalize Glu, GABA, and Gly. Furthermore, amacrine cells in the outer two or three rows of cells are more likely to be stained by Gly than Glu, Asp, or GABA. Confirming previous studies, ganglion cells were mostly immunoreactive for Glu and Asp with fewer reactive for GABA and Gly. Strong and distinctly cellular immunoreactivity was found in both central and peripheral retina. Our findings show: 1) there is extensive colocalization of Glu, Asp, GABA, and Gly among most retinal neurons, including some cells that contain all four; 2) cells of the direct functional pathway tend to be labeled by Glu and Asp generally to the exclusion of GABA and Gly, while those of the indirect pathway tend to be labeled by GABA+ and/or Gly+ in addition to Glu+ and Asp+; 3) different cell body layers have distinct patterns of colocalization; and 4) there is no qualitative difference in staining patterns between peripheral and central retina.

Microdialysis in the mouse nucleus accumbens: a method for detection of monoamine and amino acid neurotransmitters with simultaneous assessment of locomotor activity.

Microdialysis has been extensively used to characterize the effects of drugs of abuse on extracellular levels of various neurotransmitters in nucleus accumbens (NAc) of the rat brain. However, recent advances in mouse genetics have prompted the need for studying the in vivo neurochemical correlates of drug intake in genetically engineered mice. While an earlier study has shown the feasibility of measuring monoamines in the NAc of behaving transgenic mice [I. Sillaber, A. Montkowski, R. Landgraf, N. Barden, F. Holsboer, R. Spanagel, Enhanced morphine-induced behavioural effects and dopamine release in the nucleus accumbens in a transgenic mouse model of impaired glucocorticoid (type II) receptor function: influence of long-term treatment with the antidepressant moclobemide, Neuroscience, 85 (1998) 415-425 [16] ], in this protocol we demonstrate a method for measuring both monoamine and amino neurotransmitters from the NAc of freely moving mice combined with open field locomotor activity monitoring. Mice were implanted with guide cannulae aimed at the NAc and allowed 4 days of recovery before being implanted with microdialysis probes equipped with 1-mm cuprophane membranes. On the following day, mice were placed in plexiglass chambers equipped with infrared photobeams, where microdialysis samples and locomotor activity data were collected in 10-min intervals. Immediately after collection, microdialysis samples were split into two equal aliquots for separate analysis of monoamine and amino acid neurotransmitter content. High performance liquid chromatography (HPLC) analysis revealed that norepinephrine, dopamine, serotonin, aspartate, glutamate, glycine, taurine, and gamma-aminobutyric acid (GABA) could be detected in each microdialysis sample. Thus, we have shown it is feasible to monitor extracellular levels of multiple neurotransmitters with simultaneous measurement of locomotor behavior in the mouse, making this model suitable for studying differential neurochemical and behavioral responses to drugs of abuse in genetically engineered mice.

[The role of biochemical markers in peripheral body fluids in assessment of human neurotoxicity].

To date the evaluation of chemically-induced neurotoxic effects on humans has been dependent mostly on electrophysiological measurements, neurobehavioral tests and biological exposure assessment. However, recently attempts have been made to develop biochemical parameters in peripheral body fluids which can be easily obtained from humans and which can represent markers for the same parameters in nervous tissue. The approach of this kind is logically based on the following facts: 1) Blood cells (e.g., platelets and lymphocytes) possess some characteristics of monoaminergic neurons such as the existence of storage vesicles of monoamines, membrane neurotransmitter receptors, high affinity uptake sites and neurotransmitter-related metabolizing enzymes. 2) Leakage of nerve-specific markers from nervous tissue to peripheral body fluids may occur following damages of target neuronal cells or macromolecules. 3) Quantitative and/or qualitative alterations of peripheral biochemical markers (e.g. neurotransmitter receptors) can be induced by the regulation mechanisms of neuronal, endocrinal and immunologic interactions when the nervous functions are perturbed by various exogenous or endogenous factors. Erythrocyte acetyl cholinesterase (AChE), free erythrocyte protoporphyrin (FEP), lymphocyte neurotoxicity target enzyme (NTE), blood aminolevulinic acid dehydratase (ALA-D), and carboxyhemoglobin (CO-Hb) are well-known peripheral markers of the effects induced by organophosphates (AChE, NTE), lead (FEP, ALA-D) and carbon monoxide (CO-Hb). Many studies have been made on the effects of organic solvents, heavy metals and pesticides on neurotransmission parameters in blood cells such as neurotransmitter uptake, receptor binding and enzyme activity. This paper summarizes the present knowledge on the development and clinical applications of some peripheral biochemical markers such as neurotransmission parameters in blood cells and neuronal or glial cell marker proteins in CSF, blood and urine. The role of these peripheral biochemical markers in the assessment of environmental chemically-induced human neurotoxicity is also discussed.

Simultaneous liquid chromatographic determination of seventeen of the major monoamine neurotransmitters, precursors and metabolites. II. Assessment of human brain and cerebrospinal fluid concentrations.

The optimized chromatographic method procedure presented in Part I was employed for the assessment of human brain and cerebrospinal fluid neurotransmitters levels. The optimized sample preparation and chromatographic conditions permitted a rapid (less than 25 min), sensitive and semi-automated high-performance liquid chromatographic analysis which measures all major monoamine neurotransmitters, precursors and metabolites in human brain and cerebrospinal fluid. The brain specimen was deproteinized with perchloric acid (containing Na2EDTA and sodium sulphite), the internal standard and heparin were added and the samples were sonicated, centrifuged, filtered and injected directly into the chromatographic system. Cerebrospinal fluid was handled in a similar manner except that sonication was excluded. The regional distribution of monoamine neurotransmitter concentrations in human brain and cerebrospinal fluid is presented.

Patterns of DNA distribution and neurohormone immunoreactivity in the tumour cells: tools for the histopathological assessment of gastro-intestinal carcinoids.

Modern immunohistochemical and DNA cytochemical analyses of gastro-intestinal carcinoids have yielded results that have increased our knowledge of the biological properties and the histogenesis of these theoretically and practically so fascinating kinds of neoplasm. Carcinoids in different anatomical localisations were found to show marked differences with regard to their neurohormone peptide immunoreactivity pattern and their ability to evoke clinical signs and symptoms of hormone overproduction. This can be of great help to the practising pathologist when he tries to predict the anatomical site of an unknown primary tumour from the results of this histopathological assessment of a metastatic nodule of a carcinoid. The DNA distribution pattern in the nuclei of carcinoid tumour cells is a tool in the histopathological assessment of the neoplasm that seems to be of some value in predicting the subsequent clinical course of the disease. This conclusion is based on the results of a pilot study of 8 cases of ileal carcinoids with liver and lymph node metastases. It was found that 4 cases with a rapidly progressive fatal disease had a higher proportion on non-diploid tumour cell nuclei than 4 cases still alive and at full work 5 years after the diagnosis of liver metastases. However, the number of aneuploid tumour cell nuclei was negligible in both groups.

Rat brain monoamine levels related to behavioral assessment.

Randomly selected adult, male, Sprague-Dawley rats exhibit a range of behaviors in an open field. Exploration without defecation or urination is interpreted as stable behavior. On the basis of their open field behavior we selected the five most "emotional" and five most "stable" rats from two separate groups of thirty rats. Norepinephrine (NE), dopamine (DA), and serotonin (5HT) levels were determined in brains from these ten "emotional" and ten "stable" rats. The NE levels of "emotional" rats were elevated about 60 ng/g relative to the "stable" rats. There was no difference in DA levels, but there appeared to be a trend toward elevation of 5HT levels in the "emotional" rats. These findings directly support the hypothesis that elevated central nervous system norepinephrine levels may reflect a factor which contributes to emotionality in the rat, and suggest that brain norepinephrine levels may be a biochemical mechanism which influences performance as seen with the commonly used open field behavioral test of emotionality.