Jakob Korf

Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey.

Lateral flow (immuno)assays are currently used for qualitative, semiquantitative and to some extent quantitative monitoring in resource-poor or non-laboratory environments. Applications include tests on pathogens, drugs, hormones and metabolites in biomedical, phytosanitary, veterinary, feed/food and environmental settings. We describe principles of current formats, applications, limitations and perspectives for quantitative monitoring. We illustrate the potentials and limitations of analysis with lateral flow (immuno)assays using a literature survey and a SWOT analysis (acronym for “strengths, weaknesses, opportunities, threats”). Articles referred to in this survey were searched for on MEDLINE, Scopus and in references of reviewed papers. Search terms included “immunochromatography”, “sol particle immunoassay”, “lateral flow immunoassay” and “dipstick assay”.

Lymphocytes as a neural probe: potential for studying psychiatric disorders.

There is an increasing body evidence pointing to a close integration between the central nervous system (CNS) and immunological functions with lymphocytes playing therein a central role. The authors provide arguments to consider blood lymphocytes as a convenient probe of--an albeit--limited number of cellular functions, including gene expression. The use of brain biopsies of living patients is unrealistic for biochemical investigation, therefore lymphocytes may be a convenient and accessible alternative. Numerous studies showed similarities between receptor expression and mechanisms of transduction processes of cells in the nervous system (e.g. neurons and glia) and lymphocytes. In several neuropsychiatric disorders, alteration of metabolism and cellular functions in the CNS, as well as disturbances in the main neurotransmitter and hormonal systems are concomitant with altered function and metabolism of blood lymphocytes. We summarize relevant investigations on depression, stress, Alzheimers disease (AD) and schizophrenia. New techniques such as cDNA microarray gene expression and proteomics may give clues to define molecular abnormalities in psychiatric disorders and could eventually reveal information for diagnostic and treatment purposes. Taken together, these considerations suggest that lymphocyte could reflect the metabolism of brain cells, and may be exploited as a neural and possible genetic probe in studies of psychiatric disorders.

Regional rat brain levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid: concurrent fluorometric measurement and influence of drugs.

A concurrent semi-automatic fluorometric assay technique for 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), is described. The method is based on a rapid manually performed isolation of DOPAC and HVA on small columns of Sephadex G-10 followed by an automated fluorometric detection with a continuous flow system. DOPAC and HVA were measured in the corpus striatum, nucleus accumbens and olfactory tubercle of the rat, under normal conditions and after treatment with amphetamine, apomorphine, clozapine, haloperidol, morphine, oxotremorine, pargyline, probenecid, sulpiride and thioridazine. Clozapine, morphine, sulpiride and oxotremorine induced the most pronounced rise of DA metabolites in the nucleus accumbens. Probenecid produced a DOPAC accumulation in the nucleus accumbens. Striking differences were observed between the DOPAC/HVA ratios in the different structures of control animals. The concurrent assay enables a rapid screening of the action of drugs in regional DA metabolism.

β-Amyloid neurotoxicity is mediated by a glutamate-triggered excitotoxic cascade in rat nucleus basalis

Whereas a cardinal role for β‐amyloid protein (Aβ) has been postulated as a major trigger of neuronal injury in Alzheimers disease, the pathogenic mechanism by which Aβ deranges nerve cells remains largely elusive. Here we report correlative in vitro and in vivo evidence that an excitotoxic cascade mediates Aβ neurotoxicity in the rat magnocellular nucleus basalis (MBN). In vitro application of Aβ to astrocytes elicits rapid depolarization of astroglial membranes with a concomitant inhibition of glutamate uptake. In vivo Aβ infusion by way of microdialysis in the MBN revealed peak extracellular concentrations of excitatory amino acid neurotransmitters within 20–30 min. Aβ‐triggered extracellular elevation of excitatory amino acids coincided with a significantly enhanced intracellular accumulation of Ca2+ in the Aβ injection area, as was demonstrated by 45Ca2+ autoradiography. In consequence of these acute processes delayed cell death in the MBN and persistent loss of cholinergic fibre projections to the neocortex appear as early as 3 days following the Aβ‐induced toxic insult. Such a sequence of Aβ toxicity was effectively antagonized by the N‐methyl‐d‐aspartate (NMDA) receptor ligand dizocilpine maleate (MK‐801). Moreover, Aβ toxicity in the MBN decreases with advancing age that may be associated with the age‐related loss of NMDA receptor expression in rats. In summary, the present results indicate that Aβ compromises neurons of the rat MBN via an excitotoxic pathway including astroglial depolarization, extracellular glutamate accumulation, NMDA receptor activation and an intracellular Ca2+ overload leading to cell death.

Rapid concurrent automated fluorimetric assay of noradrenaline, dopamine, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 3-methoxytyramine in milligram amounts of nervous tissue after isolation on Sephadex G10.

A semi‐automated fluorimetric assay technique for the concurrent estimations of noradrenaline, dopamine, 3, 4‐dihydroxyphenylacetic acid, homovanillic acid and 3‐methoxytyramine is described. The method is based on a rapid manually performed isolation of the catecholamines and metabolites on small columns of Sephadex G10, followed by automated fluorimetric dection in continuous flow systems. Samples containing no more that 2‐5 ng (NA, DA, DOPAC or HVA) or 10 ng 3‐MT could be reproducibly measured. The small Sephadex G10 columns has proven to be an excellent alternative to the currently used columns of alumina or ion‐exchange resins.

Turnover of acid dopamine metabolites in striatal and mesolimbic tissue of the rat brain.

The subject of this study was the disappearance of 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) from the brain of rats treated with pargyline and pargyline in combination with tropolone. The DOPAC decline was exponential after pargyline treatment, while the HVA decline became a first order elimination when in addition to monoamine oxidase, catechol-O-methyl transferase was also inhibited. The turnover of the metabolites was calculated by multiplying the steady state level by the fractional rate constant (k). The DOPAC turnover was found to be 23.3 nmol/g/h in the corpus striatum and 22.6 nmol/g/h in the mesolimbic structures. The HVA turnover was 11.2 nmol/g/h in the corpus striatum and 6.7 nmol/g/h in the mesolimbic structures. The data showed that under control conditions DOPAC is only partially O-methylated to HVA, while the formation of HVA via the methoxytyramine pathway is unlikely. DOPAC turnover therefore probably approximates dopamine turnover.

Convergent genetic modulation of the endocrine stress response involves polymorphic variations of 5-HTT, COMT and MAOA

Highly prevalent stress-related disorders such as major depression (MD) are characterised by a dysregulation of the neuroendocrine system. Although heritability for these disorders is high, the role of genes in the underlying pathophysiology is poorly understood. Here, we show that polymorphic variations in genes coding for serotonin transporter (5-HTT), catechol-O-methyl transferase (COMT) and monoamine oxidase A (MAOA) as well as sex differences influence the regulation of hypothalamic–pituitary–adrenal (HPA)-axis response to acute psychological and endocrine challenges. In our sample, the effects of COMT on the release of adrenocorticotrophin hormone (ACTH) depend on the presence of the low-expression MAOA variant in the same individual. By including individuals varying in their degree of susceptibility to MD, we showed evidence of interactions between 5-HTT and MD susceptibility in baseline cortisol, and between MAOA and MD susceptibility in baseline ACTH measures, indicating a role for these genotypes in stable-state endocrine regulation. Collectively, these results indicate that the simultaneous investigation of multiple monoaminergic genes in interaction with gender have to be measured to understand the endocrine regulation of stress. These findings point towards a genetic susceptibility to stress-related disorders.

Extracellular lactic acid as an indicator of brain metabolism: continuous on-line measurement in conscious, freely moving rats with intrastriatal dialysis

Lactic acid was measured continuously in the dialysis perfusate emerging from the striatum of conscious, freely moving rats. The continuous measurement utilized a specific enzymatic/fluorometric detector that provided temporal information about the changes in the concentration of lactate in extracellular fluid (ECF). The level of lactate in extracellular fluid was found to be directly linked to local cellular metabolism. Inhibition of glycolysis with 2-deoxyglucose decreased the ECF level of lactate, whereas increased lactate production was observed after uncoupling mitochondrial electron transport with 2,4-dinitrophenol. A transient increase in the extracellular level of lactate was found after neuronal stimulation (e.g., electroconvulsive shock or local administration of kainic acid). The response to electroconvulsive shock could be attenuated by inhibiting the electrical activity of neurons with tetrodotoxin. Thus, this system is of providing novel information about transient changes in the extracellular concentration of lactic acid in real time, and these changes can be related to changes in metabolism and neuronal activity.

Fluorimetric determination of 5-hydroxyindoleacetic acid in human urine and cerebrospinal fluid

Abstract A simple fluorimetric determination of 5-hydroxyindolacetic acid in human urine and cerebrospinal fluid, based on fluorophore formation with o -phthalaldehyde, is described. Normal excretion values of 5-hydroxyindoleacetic acid of 5 persons were 1.8–4.5 mg/24 h. Concentration of 5-HIAA in cerebrospinal fluid of 5 patients of a neurological clinic ranged from 0.005 to 0.03 μg/ml. Spectrum analysis and thin-layer chromatography proved that there was no difference between the data of authentic 5-HIAA and those of the urine extract. No interfering compounds could be detected on a thin-layer chromatogram.

Neuroanatomy of Cardiac Activity-regulating Circuitry: A Transneuronal Retrograde Viral Labelling Study in the Rat

The anatomy of cardiac activity‐regulating circuitry was studied with retrograde transneuronal viral labelling after pseudorabies virus injections into different parts of the rat heart. Transection of the spinal cord at Th1 was used to reveal selectively the parasympathetic neuronal networks. Virus‐labelled sympathetic preganglionic cells were found in the Th1‐Th7 thoracic intermediolateral cell groups, with some additional infections at Th8‐Thll after inoculations of the ventricular myocardium. After ventricular injections the thoracic spinal labelling pattern was bilateral and after right atrial infection it was contralateral. Approximately 20% of the parasympathetic preganglionic cells were located in the dorsal motor vagus nucleus; the rest occupied positions in the peri‐ambiguus area ventrolateral to the nucleus ambiguus. Here and in the ventrolateral reticular formation myocardiotopy was found. Supraspinal transneuronal infections were bilateral, showed no apparent side dominance and were found in the nucleus of the solitary tract, the area postrema, the raphe nuclei, the A5 group, the parabrachial region, the periaqueductal grey, the hypothalamus, the amygdala and the cortex, in particular the anterior cingulate, the frontal, prelimbic, infralimbic and insular cortices. Spinal transections at Th1 reduced the number of labelled cells, gave a right side labelling dominance and affected the infection patterns in the ventrolateral reticular area, the raphe nuclei, the periaqueductal grey matter, the perifornical and retrochiasmatic area and the rostra1 parts of the insular cortex. The latter structures are linked selectively to the sympathetic innervation of the heart. The anatomical and functional aspects of these findings are discussed in relation to the autonomic control of heart activity.