A small, removable microdialysis probe

A miniaturized, concentric, microdialysis probe is described. It is constructed from 36 gauge stainless steel tubing inside of 26 gauge tubing, with a cellulose hollow fiber tip 0.2 mm in diameter and 2 mm long. It has a 6000 molecular weight cut off that excludes enzymes but collects monoamines, their metabolites, and other small neurochemicals. In vitro tests show relative recovery rates of 5-10%. Absolute recovery measured in picograms was independent of the perfusate flow rate inside the probe. Tests in awake rats with probes in the nucleus accumbens showed stable amounts of catecholamines and metabolites collected during repeated 20 min samples. After ip amphetamine, release of dopamine in the accumbens increased from 20 to 40 pg per sample while DOPAC and HVA decreased from about 1500 to 500 pg. Tests of multiple site sampling succeeded in obtaining norepinephrine and dopamine plus three metabolites (DOPAC, HVA and 5HIAA) from four probes simultaneously in four different brain sites in each rat. Five day continuous samples or monthly intermittent samples can be obtained with this microdialysis probe.     

Effects of Chronic Restraint Stress on Feeding Behavior and on Monoamine Levels in Different Brain Structures in Rats

Monoaminergic systems are important modulators of the responses to stress. Stress may influence feeding behavior, and the involvement of monoamines in the control of food intake is well recognized. We investigated the effects induced by chronic-restraint stress, 1 h a day, for 40 days, on eating behavior and on monoamines in distinct brain structures. Increased consumption of sweet pellets, and not of peanuts, was observed. Dopamine (DA), serotonin (5–HT), and their metabolites were measured by HPLC-EC. After chronic restraint, the results observed were decreased 5–HT in hippocampus, with increased 5–HIAA/5–HT; decreased 5–HIAA levels in cortex; reduction in DA in hippocampus, and increased levels in amygdala and hypothalamus; HVA increased in cortex, as well as HVA/DA ratio, while DOPAC/DA decreased. HVA decreased in hypothalamus, as well as HVA/DA, and DOPAC/DA and HVA/DA decreased in the amygdala. These results suggest that restraint stress differentially affects the activity of central dopaminergic and serotonergic neurons, and this may be related to the effects observed in eating behavior.     

Effects of acute handling stress on cerebral monoaminergic neurotransmitters in juvenile Senegalese sole Solea senegalensis

Juvenile Senegalese sole Solea senegalensis were subjected for short periods to two different types of handling‐related stress: air exposure stress and net handling stress. The S. senegalensis were sacrificed 2 and 24 h after the stress events and the levels of serotonin (5‐HT), noradrenaline (NA), dopamine (DA) and their respective major metabolites, 5‐hydroxyindoleacetic acid (5‐HIAA), 3‐methoxy‐4‐hydroxyphenylglycol (MHPG) and 3,4‐dihydroxyphenylacetic acid (DOPAC), were measured in three brain regions (telencephalon, hypothalamus and optic tectum) and compared with those in control, non‐stressed S. senegalensis. Neither type of stress caused any significant alteration of serotoninergic activity (5‐HIAA:5‐HT ratio) or NA levels. Dopaminergic activity (DOPAC:DA ratio) was lower in stressed fish in all of the brain regions studied. For both air exposure stress and net handling stress, DA levels were significantly higher (P < 0·05) than in the control S. senegalensis. In addition, the higher DA levels after net handling stress were always significantly higher (P < 0·05) than those observed after acute air exposure stress, except in the telencephalon after 24 h. The significantly lower DOPAC:DA ratio (P < 0·05) in all of the brain regions studied was only observed in response to net handling stress.     

Simultaneous measurement of dopamine and its metabolites, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid and tryptophan in brain tissue using liquid chromatography and electrochemical detection

An assay has been developed for brain tryptophan using reverse-phase liquid chromatography with electrochemical detection. The method simultaneously assays dopamine (DA) and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). The method does not require elution from ion exchange resins. After deproteinization and centrifugation samples are injected directly onto the chromatographic column. It was found that small changes in mobile phase pH markedly influenced the retention time of tryptophan while elution of the indoleamines and catecholamines did not change. The assay of these endogenous compounds in a single injection proved not expedient but inexpensive. Values obtained using alumina and ion exchange resins yielded comparable values.     

Tolerance develops to the haloperidol-induced increase in the efflux of dopamine metabolites from the brains of unanesthetized, freely-moving rats

The lateral cerebral ventricles of freely moving rats were perfused by means of chronically implanted push-pull cannulae every second day for 2 weeks. Perfusates were analyzed for metabolites of dopamine [dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] and of 5-hydroxytryptamine [5-hydroxyindoleacetic acid (5HIAA)] using high performance liquid chromatography and an amperometric detector. Rats received daily subcutaneous injections of haloperidol (1 mg/kg) or its vehicle. After the first injection of haloperidol the concentrations of DOPAC and HVA were markedly increased while that of 5HIAA was unchanged. Complete tolerance developed to the haloperidol-induced increased efflux of dopamine metabolites by day 9, although a higher dose of haloperidol (2 mgf/kg) on day 15 was still capable of eliciting a modest increase in the efflux of DOPAC and HVA.     

Determination of Serotonin and Dopamine Metabolites in Human Brain Microdialysis and Cerebrospinal Fluid Samples by UPLC-MS/MS: Discovery of Intact Glucuronide and Sulfate Conjugates

An UPLC-MS/MS method was developed for the determination of serotonin (5-HT), dopamine (DA), their phase I metabolites 5-HIAA, DOPAC and HVA, and their sulfate and glucuronide conjugates in human brain microdialysis samples obtained from two patients with acute brain injuries, ventricular cerebrospinal fluid (CSF) samples obtained from four patients with obstructive hydrocephalus, and a lumbar CSF sample pooled mainly from patients undergoing spinal anesthesia in preparation for orthopedic surgery. The method was validated by determining the limits of detection and quantification, linearity, repeatability and specificity. The direct method enabled the analysis of the intact phase II metabolites of 5-HT and DA, without hydrolysis of the conjugates. The method also enabled the analysis of the regioisomers of the conjugates, and several intact glucuronide and sulfate conjugates were identified and quantified for the first time in the human brain microdialysis and CSF samples. We were able to show the presence of 5-HIAA sulfate, and that dopamine-3-O-sulfate predominates over dopamine-4-O-sulfate in the human brain. The quantitative results suggest that sulfonation is a more important phase II metabolism pathway than glucuronidation in the human brain.     

Rimcazole (BW 234 U) and regional monoamine concentrations in mouse brain

Rimcazole (BW 234 U), a novel agent with antipsychotic potential, was administered i.p. (50 mg/kg) to adult male ICR mice 30 minutes before brain removal. Regional HPLC analysis of central monoamine levels showed no change in the DOPAC/DA ratio for any tissue as compared with values obtained from control saline treated animals. However, the MHPG/NE ratio was increased in the septum, striatum, hypothalamus and thalamus and there was a reciprocal decrease in the 5HIAA/5HT ratio in the septum. The data are consistent with a known lack of rimcazole affinity for dopamine receptors and indicate the involvement of alternate neurotransmitter systems in the actions of this drug.     

Intracerebrally administered (6r)-l-erythro-tetrahydrobiopterin does not affect extracellular levels of dopamine and serotonin metabolites in rat striatum in vivo during measurement by brain micro-dialysis system

By the use of the brain micro-dialysis technique combined with HPLC, the changes in the extracellular levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and a serotonin(5-HT) metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were examined in the rat striatum before and after intracerebral injection of a vehicle or (6R)-l-erythro-tetrahydrobiopterin (6R-BH₄), the natural form of the cofactor for the tryrosine hydroxylase and tryptophan hydroxylase. No apparent change after the 6R-BH, treatment was found in the levels of DA, DOPAC, HVA and 5-HIAA in the striatal dialysate. In contrast, the levels of total biopterin in both the operated (dialysis probe-implanted) and unoperated striatum of 6R-BH₄-treated rats increased by 23- and 93-fold, respectively, when compared with those of the control, vehicle-treated rats. The results indicate that increased levels of the tetrahydrobiopterin cofactor may not affect the release of DA and the extracellular level of DA and 5-HT metabolites in the physiologically normal brain.     

3,4-Dihydroxyphenylethylamine and 5-Hydroxytryptamine Metabolism in the Rat: Acidic Metabolites in Cisternal Cerebrospinal Fluid Before and After Giving Probenecid

3,4-Dihydroxyphenylethylamine (DA, dopamine) and 5-hydroxytryptamine (5-HT) turnover values were determined in freely moving male rats by measuring the rates of accumulation of the acidic metabolites of the above transmitters, i.e., 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) in cisternal cerebrospinal fluid (CSF) samples after probenecid (200 mg/kg i.p.) administration. Determinations on samples before and after acid hydrolysis showed that the latter procedure was necessary for DA turnover determination. Thus whereas total (DOPAC + HVA) increased linearly with time after probenecid, free (DOPAC + HVA) did not. This was because the percentage of DOPAC + HVA in conjugated form increased with time. Determinations on a group of 28 rats during the dark (red light) period showed that cisternal amine metabolite concentrations before probenecid injection did not parallel turnover values. This was probably because individual differences in metabolite egress strongly affect the pre-probenecid values. The poor correlations between CSF tryptophan and 5-HT turnover suggested that differences of brain tryptophan concentration were not major determinants of differences of brain 5-HT metabolism within this group of normal rats. Considering that the rats were of similar weight and that the turnover values were all determined at approximately the same time of day, the three- to fourfold ranges of the turnover values are remarkable. The positive correlation between the DA and 5-HT turnovers of individual rats suggests the existence of common effects on DA and 5-HT turnover in normal rats.     

Amine Metabolites in the Lumbar Cerebrospinal Fluid of Humans with Restricted Flow of Cerebrospinal Fluid

DETERMINATION of homovanillic acid (HVA) and 5-hydroxy-indole acetic acid (5HIAA) in human lumbar cerebrospinal fluid (CSF) is becoming an important tool in the study of the metabolism in the brain of their respective precursors, dopamine and 5-hydroxytryptamine and in the interpretation of the effects of drugs on these substances. The assumption that the concentration of the acidic metabolites HVA and 5HIAA in the lumbar CSF gives a measure of the amount of turnover of the parent amines in the brain is supported by several findings. (1) Amine metabolite concentrations in the lateral ventricular CSF of the dog correlate with their concentrations in adjacent brain areas¹. (2) Peripherally administered HVA only penetrates slightly or not at all to lateral ventricular CSF in the cat² or dog³, similar results being obtained for 5HIAA in the dog⁴. (3) Drugs which alter brain amine turnover in laboratory animals also alter the concentrations of the acidic metabolites in dog³, rabbit⁵ and human⁶ CSF in the appropriate direction. (4) In Parkinsonism and in senile and presenile dementia, conditions in which there is evidence of defective turnover of amines in the brain, low concentrations of HVA and 5HIAA are found in the CSF⁷.     

Biogenic amines and their metabolites in mouse brain tissue: development, optimization and validation of an analytical HPLC method

A simple and fast HPLC method based on an isocratic, reversed-phased ion-pair with amperometric end-point detection for simultaneous measurement of noradrenergic (MHPG/NA and A), dopaminergic (DOPAC, HVA/DA) and serotonergic (5-HIAA/5-HT) compounds in mouse brain tissue was developed. In order to improve the chromatographic resolution (Rs) with an acceptable total analysis time, experimental designs for multivariate optimization of the experimental conditions were applied. The optimal conditions for the separation of the eight neurotransmitters and metabolites, as well as two internal standards, i.e., DHBA and 5-HMT, were obtained using a mixture of methanol–phosphate–citric buffer (pH 3.2, 50 mM) (9:91, v/v) containing 2 mM OSA as mobile phase at 32 °C on a microbore ALF-115 column (150 mm × 1.0 mm, 3 μm particle size) filled with porous C₁₈ silica stationary phase. In this study, a two-level fractional factorial experimental design (½ 2^K) was employed to optimize the separation and capacity factor (k′) of each molecule, leading to a good separation of all biogenic amines and their metabolites in brain tissue. A simple method for the preparation of different bio-analytical samples in phosphate–citric buffer was also developed. Results show that all molecules of interest were stabilized for at least 24 h in the matrix conditions without any antioxidants. The method was fully validated according to the requirements of SFSTP (Société Française des Sciences et Techniques Pharmaceutiques). The acceptance limits were set at ±15% of the nominal concentration. The method was found accurate over a concentration range of 4–2000 ng/ml for MHPG, 1–450 ng/ml for NA, 1–700 ng/ml for A, 1–300 ng/ml for DOPAC, 1–300 ng/ml for 5-HIAA, 1–700 ng/ml for DA, 4–2800 ng/ml for HVA and 1–350 ng/ml for 5-HT. The assay limits of detection for MHPG, NA, A, DOPAC, 5-HIAA, DA, HVA and 5-HT were 2.6, 2.8, 4.1, 0.7, 0.6, 0.8, 4.2 and 1.4 pg, respectively. It was found that the mean inter- and intra-assay relative standard deviations (RSDs) over the range of standard curve were less than 3%, the absolute and the relative recoveries were around 100%, demonstrating the high precision and accuracy, and reliability of the analytical method described to apply in routine analysis of biogenic amines and their metabolites in brain tissue.     

Analysis of rat brain microdialysate by gas chromatography—high-resolution selected-ion monitoring mass spectrometry

Gas chromatography—high-resolution selected-ion monitoring mass spectrometry was used to analyze catecholamine metabolites in rat brain microdialysate. Dialysate samples were collected in vials containing stable isotope analogues of homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG) and 5-hydroxyindoleacetic acid (5HIAA) and analyzed as their trimethylsilyl derivatives. The metabolite levels were monitored at 20-min intervals throughout the time course of the experiment, beginning immediately after surgery and implantation of the dialysis probe and ending 4 h after amphetamine treatment. The levels of HVA were observed to decrease after amphetamine treatment, while those of MHPG and 5HIAA did not change significantly.     

Regional brain monoamine levels and utilization in middle-aged rats.

Significant changes in monoamine levels and utilization were noted in certain brain regions of middle-aged Fisher 344 rats when compared with young adult controls. In the prefrontal cortex and septum, 3,4 dihydroxyphenylglycol (MHPG) and the MHPG/norepinephrine (NE) ratio were decreased. The septum also showed increases in dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) but there was a decrease in the DOPAC/DA ratio. The striatum showed an increase in the MPHG/NE ratio and an increase in DOPAC. The hippocampus and thalamus showed an increase in 5-hydroxyindoleacetic acid (5HIAA). This demonstrates that selected neurotransmitter systems in the brain are altered at an early stage of senescence. This could lead to ensuing neurological deficits.     

Concurrent Determination of Brain Dopamine and 5-Hydroxytryptamine Turnovers in Individual Freely Moving Rats Using Repeated Sampling of Cerebrospinal Fluid

5-Hydroxytryptamine (5-HT) turnover and dopamine (DA) turnover values were obtained in individual conscious rats by measuring the rates of accumulation of 5-hydroxyindoleacetic acid (5-HIAA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in cisternal CSF samples taken from each rat at 0, 30, and 60 min after probenecid (200 mg/kg i.p.) administration. In a separate experiment, 5-HT and DA turnover values were determined in CSF, striatum, and rest of brain of groups of rats killed 0, 30, or 60 min after probenecid. Whole brain turnover values were calculated from striatal and rest of brain values. Mean turnover values using CSF were comparable with both procedures. DA turnover values were greater when based on total (i.e., free + conjugated) DA metabolites than when based on free metabolites. After partial inhibition of monoamine synthesis with the decarboxylase inhibitor DL-alpha- monofluoromethyl -DOPA ( MFMD , 100 mg/kg p.o.) DA and 5-HT turnover values were comparably reduced in whole brain, rest of brain, and CSF but more markedly reduced in the striatum. Mean DA and 5-HT turnover values obtained using CSF were similar with probenecid doses over the range 150-250 mg/kg i.p. but were variable when repeatedly determined in the same rats after administration of 200 mg/kg probenecid. Results in general show that the CSF procedure may be used to determine concurrently both 5-HT and DA turnover (when estimated from the sum of total but not free metabolites) and that it provides a good index of whole brain turnover of these transmitters in the conscious individual rat.     

Gradient liquid chromatography of leucine-enkephalin peptide and its metabolites with electrochemical detection using highly boron-doped diamond electrode

Boron-doped diamond thin film (BDD) electrodes have been used to study the oxidation reactions and to detect leucine-enkephalinamide (LEA) and its metabolites, tyrosine (T), tyrosyl-alanine (TA), tyrosyl-alanine-glycine (TAG) and leucine-enkephalin (LE) using cyclic voltammetry (CV), flow-injection analysis (FIA), and gradient liquid chromatography (LC) with amperometric detection. At diamond electrodes, well-defined and highly reproducible cyclic voltammograms were obtained with signal-to-background (S/B) ratios 5-10 times higher than those observed for glassy carbon (GC) electrodes. The analytical peaks of LC for LEA and its metabolites were well resolved. No deactivation of BDD electrodes was found after several experiments with standard as well as plasma samples, indicating high stability of the electrode. Calibration curves were linear over a wide range from 0.06 to 30 microM with regression coefficients of 0.999 for all compounds. The limits of detection obtained based on a signal-to-noise ratio of 3:1 were 3, 2.2, 2.7, 20 and 11 nM for T, TA, TAG, LE and LEA, respectively. These values were at least one order lower than those obtained at GC electrodes, which has given limits of detection of 22.88, 20.64, 89.57, 116.04 and 75.67 for T, TA, TAG, LE and LEA, respectively. Application of this method to real samples was demonstrated and validated using rabbit serum samples. This work shows the promising use of conducting diamond as an amperometric detector in gradient LC, especially for the analysis of enkephalinamide and its metabolites.     

Rat brain monoamine and serotonin S2 receptor changes during pregnancy

The concentrations of noradrenaline (NA), dopamine (DA), serotonin (5-HT), and their metabolites were determined in 5 brain areas of non-pregnant, 15 and 20 day pregnant and 4 day post-partum rats. Striatal 5-HT content was significantly lower in 15 and 20 day pregnant rats than in estrous controls. A significant decrease in striatal and frontal cortex 5-hydroxyindole-3-acetic acid (5-HIAA) concentration was observed in 15 day pregnant rats. Significant increases in hypothalamic and hippocampal NA levels were observed at 4 days post-partum. Frontal cortex serotonin S2 receptorKd was reduced in 4 day post-partum rats. There was no significant change in S2 receptorB _max during pregnancy. Levels of progesterone were negatively correlated with striatal DA, homovanillic acid (HVA), 5-HT, and 5-HIAA levels, hypothalamic DA, hippocampal 5-HT, and frontal cortex 5-HIAA values as well as striatal HVA to DA, and HVA to 3,4-dihydroxyphenylacetic acid (DOPAC) ratios and amygdaloid HVA to DOPAC ratios. The limbic neurotransmitter changes might possibly contribute to mood changes which occur during pregnancy and post-partum.     

Serotonin,dopamine, noradrenaline and their metabolites: Levels in the brain of the house cricket (Acheta domesticus L.) during a 24-hour period and after administration of quipazine—a 5-HT2 receptor agonist

1. The levels of 5-HT, DA, NA and DA metabolites (NADA, DOPAC) measured by HPLC (with electrochemical detection) in the brain of the house cricket did not change over a 24-hr period. The level of 5-HIAA, a 5-HT metabolite, was below the limit of detection.2. The 5-HT and DOPAC levels decreased and NADA increased after quipazine injection but DA and NA levels did not change after it.3. [³H]Ketanserin was used to identify serotonin receptors bound to sites in the house cricket brain with a K_d of 5 nM and a concentration of B_max 180 fmol/mg protein.     

The concentration of dopamine, 5-hydroxytryptamine,and some of their acid metabolites in the brain of genetically diabetic rats

This is a report of the concentrations of DA, DOPAC, HVA, 5-HT, and 5-HIAA in the brain ofspontaneously diabetic male Wistar rats. These rats showed a marked increase in blood and urine glucose, polydipsia, polyuria, and weight loss that had an onset 11–23 days earlier. Controls were litter mates with no hyperglycemia, glucosuria or weight reduction. Thespontaneously diabetic rats showed a significant reduction of DOPAC in the striatum, and DOPAC, HVA, and 5HIAA in the olfactory tubercles (to 69, 61, 62, and 65% of their respective controls). No changes were found in the concentrations of DA or 5-HT. Thus thespontaneously diabetic rats showed a marked reduction in striatal and mesolimbic DA and mesolimbic 5-HT metabolism. This reduction in metabolism could be the consequence of a reduction in the formation of DA and 5-HT.     

Valeriana wallichii root extract improves sleep quality and modulates brain monoamine level in rats

The present study was performed to investigate the effects of Valeriana wallichi (VW) aqueous root extract on sleep-wake profile and level of brain monoamines on Sprague-Dawley rats. Electrodes and transmitters were implanted to record EEG and EMG in freely moving condition and the changes were recorded telemetrically after oral administration of VW in the doses of 100, 200 and 300 mg/kg body weight. Sleep latency was decreased and duration of non-rapid eye movement (NREM) sleep was increased in a dose dependent manner. A significant decrease of sleep latency and duration of wakefulness were observed with VW at doses of 200 and 300 mg/kg. Duration of NREM sleep as well as duration of total sleep was increased significantly after treatment with VW at the doses of 200 and 300 mg/kg. VW also increased EEG slow wave activity during NREM sleep at the doses of 200 and 300 mg/kg. Level of norepinephrine (NE), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT) and hydroxy indole acetic acid (HIAA) were measured in frontal cortex and brain stem after VW treatment at the dose of 200mg/kg. NE and 5HT level were decreased significantly in both frontal cortex and brain stem. DA and HIAA level significantly decreased only in cortex. DOPAC level was not changed in any brain region studied. In conclusion it can be said that VW water extract has a sleep quality improving effect which may be dependent upon levels of monoamines in cortex and brainstem.     

Phencyclidine (PCP) injected in the nucleus accumbens increases extracellular dopamine and serotonin as measured by microdialysis

Phencyclidine (PCP; 20 micrograms in 0.5 microliter) was tested by local brain injection for neurochemical effects in the nucleus accumbens and striatum of rats. Changes in dopamine turnover could not be detected in postmortem tissue assays. In contrast, extracellular levels of dopamine significantly increased as measured by microdialysis in freely moving animals. PCP also increased extracellular levels of serotonin and decreased 3,4-dihydroxyphenylacetic acid (DOPAC), but did not change homovanillic acid (HVA) or 5-hydroxyindoleacetic acid (5HIAA). Microdialysis suggests that PCP acts in some dopamine terminal regions to increase extracellular dopamine and serotonin.