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Determination of 5-hydroxytryptophan, serotonin and 5-hydroxyindoleacetic acid in rat and human brain and biological fluids by reversed-phase high-performance liquid chromatography with electrochemical detection
Abstract
A rapid and sensitive method for the concurrent determination of 5-hydroxytryptophan, serotonin and 5-hydroxyindole acetic acid by reversed-phase high-performance liquid chromatography with electrochemical detection has been developed. The separation of the indolic compounds was achieved using a phosphate-citric acid eluent containing 5% methanol. Detection limits in the low picogram range were found. The method has been applied to the determination of the indolic compounds in rat and human brain tissues, as well as in human plasma and cerebrospinal fluid. Tissue and plasma preparation required only deproteinization before chromatography, while cerebrospinal fluid was directly applied to the column.
... The levels of Trp, 5-HTP, 5-HT, 5-HIAA, NE, DHPG, Tyr, L-DOPA, DA and DOPAC were measured by high performance liquid chromatography (HPLC) with electrochemical detection as described previously (Semerdjian-Rouquier et al., 1981). ...
... The probe was perfused with an artificial cerebrospinal fluid (ACSF) containing 145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl 2 /2H 2 O, 1.0 mM MgCl 2 /6H 2 O, 2.33 mM Na 2 HPO 4 , 0.45 mM NaH 2 PO 4 (pH 7.4) at a flow rate of 1 l/min by using a microinjection pump (CMA-100, CMA/Microdialysis) overnight and changed to 2 l/min with a similar ACSF solution containing 1 M of the 5-HT uptake inhibitor citalopram 2 h before the start of experiment. Serial samples were collected at 20 min intervals and extracellular 5-HT was analyzed by HPLC with electrochemical detection (Semerdjian-Rouquier et al., 1981). The average concentration of three stable fractions obtained before drug administration was defined as the 100% control value. ...
SR58611A is a selective beta(3)-adrenoceptor (Adrb3) agonist which has demonstrated antidepressant and anxiolytic properties in rodents. The present study confirmed the detection of Adrb3 mRNA transcript in rodent brain sub-regions and evaluated the effect of SR58611A on serotonergic and noradrenergic transmission in rats and mice in an attempt to elucidate the mechanism(s) underlying these properties. SR58611A (3 and 10 mg/kg, p.o.) increased the synthesis of 5-HT and tryptophan (Trp) levels in several rodent brain areas (cortex, hippocampus, hypothalamus, striatum). Moreover, SR58611A (10 mg/kg, p.o.) increased the release of 5-HT assessed by in vivo microdialysis in rat prefrontal cortex. Systemic (3 mg/kg, i.v.) or chronic administration of SR58611A (10 mg/kg, p.o.), in contrast to fluoxetine (15 mg/kg, p.o.), did not modify the activity of serotonergic neurons in the rat dorsal raphe nucleus. The increase in 5-HT synthesis induced by SR58611A was not observed in Adrb3s knockout mice, suggesting a selective involvement of Adrb3s in this effect. SR58611A (3 and 10 mg/kg, p.o.) did not modify norepinephrine synthesis and metabolism but increased its release in rat brain. Repeated administration of SR58611A (10 mg/kg, p.o.) did not modify basal norepinephrine release in rat prefrontal cortex whereas it prevented its tail-pinch stress-induced enhancement similarly to reboxetine (15 mg/kg, p.o.). Finally SR58611A increased the firing rate of noradrenergic neurons in the rat locus coeruleus following systemic (3 mg/kg, i.v.) or local (0.01 and 1 microM) but not chronic (10 mg/kg, p.o.) administration. These results suggest that the anxiolytic- and antidepressant-like activities of SR58611A involve an increase of brain serotonergic and noradrenergic neurotransmissions, triggered by activation of Adrb3s.
... Many experimental methods having been developed for the analysis of these brain monoamines like fluorometric detection, chemiluminiscence detection, gas chromatography (GC), capillary zone electrophoresis, liquid chromatography with mass spectrometry (LCMS), and high performance liquid chromatography (HPLC) with electrochemical detector (ECD) (Lunte & O'Shea, 1994;Saha et al., 2002;Semerdjian-Rouquier et al., 1981). The estimation of monoamines by HPLC with ECD is the most widely used method (Cooper et al., 1994;Kulkarni & Dhir, 2008), but due to sudden noise problems, it is not preferable for routine analysis (Yoshitake et al., 2004;Carrera et al., 2007). ...
Norepinephrine and serotonin are two important neurotransmitters whose variations in brain are reported to be associated with many common neuropsychiatric disorders. Yet, relevant literature on estimation of monoamines in biological samples using HPLC-UV is limited.
The present study involves the development of a simultaneous HPLC-UV method for estimation of norepinephrine and serotonin along with optimization of the sample preparation technique.
Chromatographic separation was achieved by injecting 20 µL of the sample after extraction into Quaternary pump HPLC equipped with C18 column using 0.05% formic acid and acetonitrile (90:10, v/v) as the mobile phase with 1 mL min(-1) flow rate. The developed method was validated as per the ICH guidelines in terms of linearity, accuracy, repeatability, precision, and robustness.
The method showed a wide range of linearity (50-4000 and 31.25-4000 ng mL(-1) for norepinephrine and serotonin, respectively). The recovery was found to be in the range of 86.04-89.01% and 86.43-89.61% for norepinephrine and serotonin, respectively. The results showed low value of %RSD for repeatability, intra and inter-day precision, and robustness studies. Four different methods were used for the extraction of these neurotransmitters and the best one with maximum recovery was ascertained.
Here, we developed and validated a simple, accurate, and reliable method for the estimation of norepinephrine and serotonin in mice brain samples using HPLC-UV. The method was successfully applied to quantify these neurotransmitters in mice brain extracted by optimized sample preparation technique.
... Estimation of catecholamines: Adrenaline and noradrenaline levels in plasma and brain of rats of all the experimental groups were determined employing HPLC method of Semerdjian-Rouquier et al. [16] using high performance liquid chromatogram (Shimadzu Corporation, Japan) having C 18 reversed phase column (125 mm × 5 mm I.D.) and L-ECD6A electrochemical detector. ...
Present study was undertaken to elucidate the ameliorating potential of Withania somnifera root extract (WRE) against lead-induced augmentation of adrenergic response in rat portal vein.
In-vitro studies were conducted on effect of lead alone and lead+WRE on rat-isolated portal vein while in-vivo studies were done in three groups of 12 rats each; Group-II and III received 0.5% lead acetate and 1.0% WRE + 0.5% lead acetate, respectively, in drinking water for 12 weeks whereas group-I served as control. Adrenaline and noradrenaline levels in brain and blood were determined by HPLC assay while vascular reactivity of portal vein to lead and WRE was determined by measuring the isometric tension.
Following in-vitro exposure, lead did not alter the contractile effect of phenylephrine. In-vivo studies revealed that contractile effect of lead on portal vein was significantly potentiated and it was antagonized by prazosin (10(-7) M) and WRE (1%). WRE treatment significantly reduced elevated blood noradrenaline (37.80%) and restored noradrenaline level in brain (39.39%) in lead-exposed animals. These values were almost comparable to the control group. But it failed to significantly affect the blood and brain adrenaline levels.
Results suggest that following pre-exposure of rats to WRE, lead-induced augmentation of alpha 1-adrenoceptors mediated response was reversed possibly by regulating catecholamine release from nerve endings. Thus, WRE may be useful in therapeutic management of lead-induced hypertension.
... After centrifugation (15,000 g for 10 min), 5-HT was assayed on 100 J.d of the superna tant by high-performance liquid chromatography (HPLC) with electrochemical detection. according to Semerdjian Rouquier et al. (1981). The limit of sensitivity of the assay (signal-to-noise ratio of 2) was 10 pg. ...
The levels of noradrenaline, neuropeptide Y, 5-hydroxytryptamine, and substance P were measured and compared between the large arteries of the circle of Willis and the small cerebral vessels of the pia mater in the rat, rabbit, cat, and monkey. In all species, noradrenaline and neuropeptide Y concentrations were greater in the larger arteries than in small pial vessels. Noradrenaline concentrations were dramatically reduced following cervical sympathectomy, with the extent of diminution differing greatly in the various species; the effects of cervical ganglionectomy on neuropeptide Y concentrations were less pronounced. 5-Hydroxytryptamine concentrations in rats, cats, and rabbits were significantly greater in the small pial vessels, although measurable concentrations existed in the circle of Willis. In cats and monkeys, substance P was found in major arteries, but was not detectable at the level of the small pial vessels. The differences in the regional distribution of the various neurotransmitter candidates in the cerebrovascular bed may reflect their physiological significance.
The use of ultrasonic waves for the preparation of inclusion complexes in pharmaceutical industries has increased, owing to their versatility, promising nondestructive capability, and short reaction time. 5-Hydroxytryptophan (5-HTP), a precursor of serotonin, is used therapeutically, in the clinical setting, for psychiatric disorders such as anxiety and depression. In this study, 5-HTP is used as a guest molecule to prepare β-cyclodextrins (CDs; β-CD, HPβ-CD, and SBEβ-CD) inclusion complexes with the assistance of the ultrasonic method. The solubility of 5-HTP increases linearly as the concentration of CDs increased, confirming the 1:1 stoichiometry of the complex, obtained using UV-visible and fluorescence spectroscopy. The structural characteristics of the solid inclusion complexes are analyzed using various spectroscopic and microscopic techniques such as FTIR spectroscopy, PXRD, TGA, and FE-SEM. Molecular modeling elucidates the most stable inclusion model and phase solubility studies indicate that 5-HTP and SBEβ-CD form a 1:1 inclusion complex with an apparent stability constant of 1592.5 M−1. Furthermore, in vitro cytotoxicity studies show that the CDs:5-HTP complex has a better antitumor efficacy than pure 5-HTP. Thus, ultrasonication can be utilized as an advanced, time-saving, and cost-effective method for the generation of CD:5-HTP inclusion complexes, resulting in the potential development of anticancer drugs.
From an anatomical point of view, the serotonin axonal varicosities in the locus coeruleus have been defined as nonsynaptic terminals originating from the raphe dorsalis, raphe centralis, and raphe pontis. Electrolytic or chemical destruction of these serotoninergic afferents has produced pronounced increases in the activity of the noradrenaline biosynthetic enzyme, tyrosine hydroxylase, in the locus coeruleus and in the noradrenaline metabolite DOPEG in the terminal field of the hippocampus and cerebral cortex. A good correlation has been observed between the depletion of serotonin and the increase in tyrosine hydroxylase activity in the locus coeruleus after chemical denervation of serotoninergic terminals. Electrical stimulation of serotonin cell bodies has increased noradrenaline levels in the locus coeruleus and cerebral cortex. Altogether these data suggest that the function of the ascending noradrenergic system originating from the locus coeruleus might be largely dependent on its interaction with serotonin-containing neurons in the raphe system.
The effects of α,β-methylene-adenosine triphosphate, (α,β-methylene ATP, a P2-receptor desensitising agent) have been evaluated on vasoconstrictor responses elicited by exogenous agonists or electrical field stimulation in isolated perfused SHR or WKY tail arteries and on tritium release elicited by electrical field stimulation in SHR-tail arteries pre-labeled with 3H-noradrenaline.
Exposure to α,β-methylene ATP (0.1 μmol/l) significantly inhibited vasoconstrictor responses to electrical field stimulation in SHR tail arteries. These inhibitory effects were not further increased at a higher concentration of α,β-methylene ATP (1 μmol/l). In WKY tail arteries, α,β-methylene ATP (1 μmol/l) failed to significantly inhibit vasoconstrictor responses to electrical stimulation.
In SHR tail arteries prelabelled with 3H-noradrenaline, α,β-methyleneATP (1 μmol/l) did not inhibit the stimulation evoked release of tritium. However, at this concentration, α,β-methylene ATP significantly antagonized the vasoconstrictor responses of SHR tail arteries induced by exogenous ATP (1 μmol/l), β,γ-methylene ATP (30 μmol/l), a stable agonist at P2-receptors, or 60 mmol/l KCl. These effects of α,β-methylene ATP on contractile responses to KCl were not observed in WKY-tail arteries.
In tail arteries obtained from reserpine pretreated SHR, despite a 85–95% decrease in endogenous noradrenaline tissue content, the vasoconstrictor responses induced by periarterial field stimulation were greatly diminished, but not abolished. These residual responses to periarterial field stimulation were not antagonized by prazosin (0.1 μmol/l), but were practically abolished by the addition of α,β-methylene ATP (1 μmol/l).
In tail arteries from WKY rats pretreated with reserpine, exposure to prazosin (0.1 μmol/l) further reduced the residual responses elicited by electrical field stimulation. In these WKY-tail arteries, addition of α,β-methylene ATP (1 μmol/l) did not further inhibit the remaining vasoconstrictor response obtained in the presence of prazosin.
While our results suggest a significantly greater cotransmitter role for ATP with noradrenaline in tail arteries of SHR compared with control normotensive WKY rats, additional effects of α,β-methylene ATP not involving P2 receptors cannot be entirely excluded.
A high-performance liquid chromatographic (HPLC) method was used to analyze a mixture of plant hormones—intermediates of the naturally occurring phytohormone, indole-3-acetic acid. The determination was performed on an analytical column (A, 250 × 2 mm) preceded by a short (50 × 2 mm) precolumn, and on a semipreparative column (B, 500 × 8 mm). The columns were filled with LiChrosorb RP- 18 and eluted with water-acetic acid-ethanol (79.2:0.8:20) at flow-rates of 40 ml/h (A) and 180 ml/h (B). Detection was done by a UV detector (280 nm) and a fluorometric detector (excitation, 280 nm; emission, 360 nm) connected in series. The detection limit for indole derivatives was 5-20 ng with UV detection and tenths to several ng with the fluorometer. Qualitative analysis was done chromatographically and confirmed by UV spectroscopy and mass spectrometry. The substances were determined by the methods of standard addition and internal normalization. The standard deviation of a determination was ⩽4.2% (relative). The HPLC analysis was used to determine growth hormones of the indole type in extracts from cultures of soil bacteria.
Powerful separation methods developed during the past few decades, in particular glass capillary gas chromatography (GC) and high performance liquid chromatography (HPLC), can be combined with sensitive methods of detection such as mass spectrometry (MS) to provide new insights into metabolic processes. Research in a field that was long thought to be exhausted has thus been revitalized. New perspective have been opened up for studying the metabolism of foods, spices and flavorings, drugs, and compounds produced in the body as well as for establishing the way in which the organism rids itself of harmful substances or how and when these compounds can interfere with the metabolic processes occurring in the body.
Although yohimbine has long been known to increase arousal, reactivity and anxiety in animals and humans, little is known about the behavioural effects of more selective a2-adrenoceptor antagonists such as idazoxan. In a recent experiment, however, it was found that in rats both yohimbine and idazoxan increased low rates of lever pressing, an effect also produced by amphetamine. The purpose of the present study was to investigate further the effects of yohimbine and idazoxan in comparison with those of d-amphetamine on the operant behaviour of rats. In rats trained to press a lever on a FI 60s schedule to obtain food both yohimbine and idazoxan increased response rates, although the effect of yohimbine was considerably greater than that of idazoxan. Lower doses of d-amphetamine had no consistent effect on overall rates of responding whereas a higher dose suppressed responding. Characteristically, d-amphetamine increased responding during early portions of the intervals and decreased responding during the final portions. Idazoxan and yohimbine tended to increase responding throughout the intervals except immediately after reinforcement. When idazoxan was administered in combination with prazosin FI response rates were markedly decreased. Administration of DSP4 did not alter the response rate-increasing effects of either yohimbine or idazoxan. In rats trained to discriminate d-amphetamine from saline both idazoxan and yohimbine gave rise to responding on the saline associated lever. Combination of idazoxan with d-amphetamine did not antagonise the amphetamine cue but produced substantial reductions in response rates, probably due to toxicity. These results confirm previous findings that both idazoxan and yohimbine have behavioural stimulant effects but do not clarify the mechanisms involved. It is clear, however, that the behavioural actions of these a2-adrenoceptor antagonists have little in common with those of the psychomotor stimulant amphetamine.
A liquid chromatographic analysis with electrochemical detection is proposed for simultaneous determination of four indols (i.e. tryptophan, 5-hydroxytryptophan, serotonin and 5-hydroxyindoleacetic acid) in various rat brain areas. Optimal elution conditions have been determined by studying chromatographic parameters : pH, buffer salt, counter-ion, organic modifier. Eventual interferences with the catecholaminergic compounds (i.e. norepinephrine, dopamine and their major metabolites) known to be detectable in the same conditions, have been eliminated. The specificity of the four indol detection was demonstrated by chromatographic, electrochemical and pharmacological proofs.
SL 81.0385 (4-[(2-naphthalenyl)methoxy]piperidine) is a potent competitive inhibitor of serotonin uptake into rat hypothalamic synaptosomes and human platelets (IC50 values of 18 and 13 nM, respectively), which exhibits a high selectivity for inhibiting serotonin uptake relative to noradrenaline or dopamine uptake in vitro. SL 81.0385 also exhibits potent serotonin uptake inhibiting properties in vivo as demonstrated by its ability to antagonize the p-chloroamphetamine-induced depletion of rat cerebral serotonin levels (ED50 = 2.5 mg/kg i.p.). Similarly, SL 81.0385 is a potent inhibitor of 3H-imipramine and 3H-paroxetine binding to the serotonin transporter complex in rat cortical membranes (Ki values of 0.5 and 3.2 nM, respectively), but it exhibits a much lesser affinity for radioligand binding to the noradrenaline, adrenaline, and dopamine transporters. The selectivity of SL 81.0385 for serotonergic systems is also demonstrated by its effects on cerebral monoamine metabolism. Indeed, SL 81.0385 decreases, ex vivo, endogenous 5-hydroxyindoleacetic acid (5-HIAA) levels in the rat striatum, and, in vivo, extracellular 5-HIAA concentrations (as measured by differential pulse voltammetry) in the rat cerebral cortex. In contrast, SL 81.0385 fails to decrease striatal levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid and hypothalamic levels of total, 3,4-dihydroxyphenylethyleneglycol. SL 81.0385 is devoid of inhibitory activity toward monoamine oxidase and has no affinity for cerebral muscarinic, H1 histaminergic, α1-, α2-, and β-adrenergic, D2 dopaminergic, and benzodiazepine receptors, and it possesses only weak affinity for 5-HT2 receptors. The present results indicate that SL 81.0385 is a highly potent and selective inhibitor of serotonin uptake, chemically unrelated to the tricyclics, and it should find application in the treatment of depression, obesity, and alcoholism.
Die in den letzten Jahrzehnten entwickelten potenten Trennverfahren, insbesondere die Glaskapillargaschromatographie (GC) und die Flüssigkeitschromatographie (High Performance Liquid Chromatography, HPLC) können uns in Kombination mit empfindlichen Nachweismethoden wie der Massenspektrometrie (MS) neue Einblicke in Stoffwechselprozesse gewähren. Damit wird die Forschung auf einem Gebiet, das lange Zeit als nahezu abgeschlossen galt, neu belebt. Neue Perspektiven eröffnen sich auch für Untersuchungen über die Metabolisierung von Nahrungs-, Genuß- und Arzneimitteln im Körper sowie über die Art, wie sich der Organismus schädlicher Stoffe entledigt und wie und wann diese Verbindungen in das körpereigene Stoffwechselgeschehen eingreifen.
It is commonly believed today that approximately 40% of autistic children have elevated blood serotonin. The phenomenon has stimulated investigators to attempt to manipulate and observe blood serotonin levels and concurrent clinical phenomena in these persons. In 1971 Ritvo et al. described attempts to lower blood serotonin levels by L-DOPA adminstration and to observe associated clinical and physiological changes. They showed that the medication could indeed produce a decrease in blood serotonin and its metabolites, but they detected no consistent clinical changes in the subjects studied. Fenfluramine, an anorexigenic amine "diet drug" first marketed in the 1960s, was found to alter blood serotonin levels in animals and 5-HIAA levels in man. In 1982 Geller, Ritvo, Freeman, and Yuwiler administered fenfluramine to three autistic patients with high blood serotonin. They showed that fenfluramine did in fact reduce blood serotonin, and that associated with this reduction were substantial improvements in performance on IQ tests and in the Ward Symptom Rating Scale. Clinical observations during the treatment period also indicated increased social respon
This communication reports the HPLC separation and quantitative ECD assay of dopamine (DA) and its metabolites DOPAC and HVA, 5-HT and its metabolite 5-HIAA and the noradrenaline metabolites MHPG and VMA, in samples of rat brain extracts and human CSF. The separation is carried out by reversed-phase with a methanolic phosphate/citrate buffer as mobile phase. Response is linear within 10pg-20 ng. Rat brain homogenates of cortex plus striatum were centrifuged and 10–20 l aliquots injected in the column. CSF samples were directly injected without any further manipulation. The method has been applied to the study of the possible neuromodulating role of T on the catecholaminergic and serotonergic transmission. For this purpose rats are injected intraperitoneally (ip) with T (150 mg/kg) and killed after 30 min. Relative to control rats, the results show that for n=12, T does not affect the basal level of DA and DOPAC whereas HVA increases a 99.3% and 5HT and 5HIAA show variations of 23% and — 4.1%, respectively. Aside from the fall of 5HIAA, it is interesting to note that the turnover rate of 5HT decreases, which might prove of functional significance.
Neurotransmitter levels are best measured in the cerebrospinal fluid (CSF), but that requires an invasive procedure.
Samples were collected from humans and rats. Eighteen women age 38-51years with fibromyalgia provided samples of CSF, plasma, platelets, and urine. Samples of CSF, plasma, platelets, and urine were also collected from Sprague-Dawley rats, adult male, 6months old. One group of rats was treated with p-chlorophenylalanine to decrease their levels of serotonin, and another group of rats was treated with amphetamine to increase their levels of serotonin. Methodological improvements include: 1) the use of siliconized glassware, plasticware, and tubing to prevent adsorption of serotonin, 2) the extraction of serotonin from the CSF, plasma, and platelets, 3) repeated washing of the platelets with an improved buffer, and 4) early morning sample collection. HPLC/MS was used to measure serotonin after extraction.
For serotonin, the new method of measuring platelet levels resulted in a very high correlation with levels of serotonin in CSF in rats (r=0.97) and humans (r=0.97). There were lower correlations of levels of serotonin in CSF with levels in plasma (r=0.77 for rats and r=0.57 in humans) and urine (r=0.67 in rats and r=0.62 in humans).
This method of measuring serotonin levels in platelets results in a very strong correlation with levels in CSF, so in most cases platelet measurements will be preferable since it is much less invasive to collect. Levels of serotonin in plasma and urine are significantly but less strongly correlated with levels in CSF.
A new HPLC technique for the analysis of picomolar amounts of serotonin (5HT) in plasma and cerebrospinal fluid (CSF) is described. Bufotenin is used as internal standard. Detection is achieved electrochemically or fluorimetrically. The detection limit can be estimated as 50 pg 5HT/mL of either fluid (0.3 picomolar). The method is used to characterize a non-particulate pool of 5HT which is clearly distinct of the platelet pool. Administration of parachlorophenylalanine (PCPA) 300 mg/kg to rats leads to a 90% reduction in the plasmatic pool whereas platelet 5HT is only slightly decreased (3rd day after PCPA) or even increased (7th day after PCPA). Human concentration (n=15) of 5HT in plasma is 2.6 ± 0.9 ng/mL (x ± S.D.). The application of the method to CSF of neurological patients reveals 5HT concentrations ranging from 93 to 962 pg/mL.
CGS 12066B is a novel pyrroloquinoxaline with selectivity for the serotonin-1B (5HT1B) recognition site as assessed by binding, biochemical and electrophysiological studies. The compound had an IC50 value of 51 nM at the 5HT1B recognition site as determined using the binding of [3H]5HT in the presence of 1 microM spiperone. At the 5HT1A receptor the compound had an IC50 value of 876 nM, providing a 5HT1A/5HT1B ratio of 17 in contrast to the putative 5HT1B selective agent trifluoromethylphenylpiperazine (TFMPP) which had a corresponding ratio of 3.6. The compound had minimal affinity for alpha 1-, alpha 2- and beta-adrenoceptors and for dopamine D-1 and D-2 receptors. CGS 12066B, in contrast to TFMPP, which was inactive, was found to inhibit dorsal raphe cell firing with an ED50 value of 358 nmol/kg i.v. The corresponding values for the 5HT1A selective agonists 8-OH-DPAT and ipsapirone were 1.3 and 33 nmol/kg. CGS 12066B was also effective in decreasing rat brain 5-HTP concentrations and inhibiting in vitro 5HT release. The data obtained indicate that CGS 12066B is a reasonably active 5HT1B site agonist, which due to its selectivity as compared to compounds such as TFMPP, will be a useful tool for evaluating the physiological role of such receptors in the mammalian CNS.
Unilateral 6-hydroxydopamine lesion of the substantia nigra reduced the volume of striatal necrosis and suppressed the increase in extracellular glutamate concentration in the striatum induced by middle cerebral artery occlusion in rats. These results indicate that the dopaminergic nigrostriatal pathway is highly involved in the vulnerability of the striatum to ischemia and suggest that glutamate-dopamine interactions may play a key role in the striatal ischemic insult.
Using in vivo microdialysis, we have examined the effects of local administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) into either the dorsal (DRN) or the median (MRN) raphe nucleus on extracellular levels of serotonin (5-HT) in the corresponding projection fields, namely striatum and ventral hippocampus in the anaesthetized rat. Local injection of 8-OH-DPAT (0.5 microgram/0.1 microliter) in the DRN reduced extracellular 5-HT levels in the striatum (-55%) and to a lesser extent in the hippocampus (-22%). When injected at the same dose in the MRN, 8-OH-DPAT caused a marked decrease in 5-HT output in the hippocampus (-41%) and had no effect in the striatum. Autoradiographic studies performed on brains from animals that received a local injection of [8H]8-OH-DPAT into either the DRN or MRN under similar experimental conditions indicated that the radioactivity remained localized within each midbrain raphe nucleus. These results confirm anatomical data demonstrating that the striatum and the ventral hippocampus receive their serotonergic innervation preferentially from the DRN and the MRN, respectively.
Based on the fact that 1-(2-methoxyphenyl)-4(4-(2-phtalimido)butyl)piperazine (NAN-190), a high-affinity ligand for 5-HT1A and alpha 1-adrenoceptors, antagonizes the behavioural effects of the 5-HT1A receptor agonist 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin), it has been suggested that this drug behaves as a 5-HT1A receptor antagonist. In the present study we examined the effects of this putative 5-HT1A receptor antagonist on rat brain serotonergic neurotransmission. In hippocampal slices of immature rats, NAN-190 but not prazosin potently antagonized (IC50 = 29 nM) the inhibitory effect of 8-OH-DPAT (1 microM) on carbachol-stimulated phosphoinositide turnover but (up to 1 microM) failed to alter the carbachol response. Similarly, NAN-190 (0.1 microM) almost totally prevented the inhibition by 8-OH-DPAT (1 microM) of forskolin-stimulated adenylate cyclase activity in adult rat hippocampal slices but, per se, was without effect on the forskolin response. These results indicate that NAN-190 is a potent antagonist at postsynaptic 5-HT1A receptors in vitro. However, NAN-190 also potently antagonized (IC50 = 0.16 nM) the stimulation by norepinephrine of phosphoinositide turnover in rat cortical slices. In this respect NAN-190 was a 250-fold more potent antagonist than prazosin (IC50 = 49 nM). Thus, in addition to its 5-HT1A receptor antagonist properties, NAN-190 has potent alpha 1-adrenoceptor blocking properties.(ABSTRACT TRUNCATED AT 250 WORDS)
The levels of noradrenaline (NA), serotonin (5-HT), and 5-hydroxyindoleacetic acid were measured by HPLC and compared between the large arteries of the circle of Willis and the small pial vessels in the rat, following either electrical stimulation of the dorsal raphe nucleus or bilateral superior cervical ganglionectomy. With electrical stimulation, the 5-HT concentrations were reduced (-48%) in the small pial vessels, but were unchanged in the major cerebral arteries. NA concentrations were dramatically reduced following cervical sympathectomy in the large arteries (-77%), though the reduction was less pronounced (-34%) in the small vessels. Sympathectomy caused a significant decrease in the 5-HT concentration of the major cerebral arteries (-33%), but was without effect on the 5-HT levels of the small pial vessels. These results show that an appreciable fraction of the perivascular 5-HT measured in the small pial and the large cerebral arteries originates from different sources.
We have studied the nature and origin of the serotonergic innervation of two distinct anatomical cerebrovascular compartments, namely, small pial vessels and major cerebral arteries, in the rat. To this end, the levels of serotonin [5-hydroxytryptamine (5-HT)] and 5-hydroxyindoleacetic acid (5-HIAA) were measured by HPLC in both cerebrovascular compartments after either bilateral sympathectomy or destruction of the ascending serotonergic pathways, which originate from the raphe nuclei. We first showed that the small pial vessel samples were not contaminated by underlying cortical tissues through the use of an immunohistochemical approach that revealed the glia limitans, the most superficial cortical layer. Superior cervical ganglionectomy caused a marked decrease in noradrenaline concentrations in major cerebral arteries (-77%), although the reduction was less pronounced (-34%) in small pial vessels. Sympathectomy decreased by 33% 5-HT concentrations in the major cerebral arteries but was without effect on 5-HT levels in the small pial vessels. Destruction of the ascending serotonergic pathways (via local administration of 5,7-dihydroxytryptamine into the ventral tegmental area) produced a dramatic fall in 5-HT and 5-HIAA concentrations in both vascular compartments. To establish the authenticity of the serotonergic innervation, the synthesis of 5-HT [as assessed by measuring the accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition] was measured in the two vascular beds under control conditions and after destruction of the ascending serotonergic pathways. The rate of accumulation of 5-HTP was higher in the small pial vessels than in major cerebral arteries, an observation that indicates an important de novo synthesis of 5-HT in small pial vessels.(ABSTRACT TRUNCATED AT 250 WORDS)
Serotonin 5-HT1A receptors have been reported to be negatively coupled to muscarinic receptor-stimulated phosphoinositide turnover in the rat hippocampus. In the present study, we have investigated further the pharmacological specificity of this negative control and attempted to elucidate the mechanism whereby 5-HT1A receptor activation inhibits the carbachol-stimulated phosphoinositide response in immature or adult rat hippocampal slices. Various 5-HT1A receptor agonists were found to inhibit carbachol (10 microM)-stimulated formation of total inositol phosphates in immature rat hippocampal slices with the following rank order of potency (IC50 values in nM): 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (11) greater than ipsapirone (20) greater than gepirone (120) greater than RU 24969 (140) greater than buspirone (560) greater than 1-(m-trifluoromethylphenyl)piperazine (1,500) greater than methysergide (5,644); selective 5-HT1B, 5-HT2, and 5-HT3 receptor agonists were inactive. The potency of the 5-HT1A receptor agonists investigated as inhibitors of the carbachol response was well correlated (r = 0.92) with their potency as inhibitors of the forskolin-stimulated adenylate cyclase in guinea pig hippocampal membranes. 8-OH-DPAT (10 microM) fully inhibited the carbachol-stimulated formation of inositol di-, tris-, and tetrakisphosphate but only partially antagonized (-40%) inositol monophosphate production. The effect of 8-OH-DPAT on carbachol-stimulated phosphoinositide turnover was not prevented by addition of tetrodotoxin (1 microM), by prior destruction of serotonergic afferents, by experimental manipulations causing an increase in cyclic AMP levels (addition of 10 microM forskolin), or by changes in membrane potential (increase in K+ concentration or addition of tetraethylammonium). Prior intrahippocampal injection of pertussis toxin also failed to alter the ability of 8-OH-DPAT to inhibit the carbachol response. Carbachol-stimulated phosphoinositide turnover in immature rat hippocampal slices was inhibited by the protein kinase C activators phorbol 12-myristate 13-acetate (10 microM) and arachidonic acid (100 microM). Moreover, the inhibitory effect of 8-OH-DPAT on the carbachol response was blocked by 10 microM quinacrine (a phospholipase A2 inhibitor) but not by BW 755C (100 microM), a cyclooxygenase and lipoxygenase inhibitor. These results collectively suggest that 5-HT1A receptor activation inhibits carbachol-stimulated phosphoinositide turnover by stimulating a phospholipase A2 coupled to 5-HT1A receptors, leading to arachidonic acid release. Arachidonic acid could in turn activate a gamma-protein kinase C with as a consequence an inhibition of carbachol-stimulated phosphoinositide turnover. This inhibition may be the consequence of a phospholipase C phosphorylation and/or a direct effect on the muscarinic receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
An improved method for the determination of serotonin in platelet-rich plasma (PRP) and platelet-poor plasma (PPP), by reversed-phase high-performance liquid chromatography with electrochemical detection and direct plasma injection, is described. The chromatographic system comprises a strong cation-exchange pre-column and a C18 analytical column. The method is selective, rapid, simple and sensitive, and offers good reproducibility and recovery. Reference values for serotonin concentrations in healthy adults (n = 10) are 31 nM for PPP and 6 nmol per 10(9) platelets for PRP. The conditions used for the preparation of PRP and PPP may influence the serotonin concentration in PRP.
The mesocorticolimbic dopaminergic neurons, orginating in the A10 dopaminergic cell group in the ventral tegmental area, are involved in the control of cognitive processes and emotional behavior. This chapter discusses the involvement of the mesocorticolimbic dopaminergic systems in emotional states. The involvement of mesocorticolimbic dopaminergic neurons in emotional behavior is supported by the demonstration that stressful conditions lead to an increase in dopamine (DA) metabolism in the corresponding DA terminal fields in the rodent. Electrolytic or neurotoxic lesions of the ventral tegmental area in the rat induce a characteristic syndrome, including hypoemotionality, locomotor hyperactivity with repetitive behavior, hypoexploratory behavior, and difficulties in suppressing previously learned responses. The lesions of dopaminergic terminals in the nucleus accumbens provoke impairment in functional processes, leading from motivation to action. Stressful conditions cause an activation of dopaminergic neurons that project to the frontal cortex and/or nucleus accumbens in the rodent. A variety of anxiogenic environmental or pharmacological stimuli shows to increase DA metabolism in the mesocortical dopaminergic system of the rat.
The present study was performed to determine the effect of a nearly complete nigrostriatal dopaminergic denervation on DARPP-32 levels in the striatum from animals and parkinsonian patients. DARPP-32 levels were estimated by in vitro phosphorylation in the presence of cAMP, or after inactivation of endogenous kinases and phosphatases, in the presence of the catalytic subunit of cAMP-dependent protein kinase. Intranigral 6-hydroxydopamine (6-OHDA) infusion in rats, or peripheral administration of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to common marmosets, did not change striatal DARPP-32 levels. Postmortem studies, carried out on brains obtained shortly after death, from patients with Parkinson disease, or from patients with progressive supranuclear palsy, showed that the levels of striatal DARPP-32 were not different from controls. These results indicate that dopaminergic striatal denervation did not modify the amount of DARPP-32 in the striatum, suggesting that the expression of DARPP-32, a protein which mediates some of the effects of dopamine in striatal neurons, is independent from the dopaminergic innervation.
The dorsal and median raphe 5-HT neurons give rise to projections that differ in axon morphology and in vulnerability to certain amphetamine derivatives. The present study was undertaken to determine if these two 5-HT systems possess different functional properties. To this end, we studied the effects of selective 5-HT1A or 5-HT1A/5-HT1B receptor agonists and of p-chloroamphetamine on extracellular levels of indoleamines, as measured by differential pulse voltammetry with extracellular levels of indoleamines, as measured by differential pulse voltammetry with electrochemically pretreated carbon fiber electrodes, in cell body and nerve terminal regions of these subsets of 5-HT neurons in the rat brain. The selective 5-HT1A agonist 8-OH-DPAT produced a gradual decrease in the height of the 300 mV oxidation peak in the dorsal raphe and in the frontal cortex, reaching a maximum of 60% 3 h after the i.v. injection of 30 micrograms/kg. However, the same dose of 8-OH-DPAT was ineffective in the median raphe and in the dentate gyrus that receives its 5-HT innervation exclusively from the median raphe. A higher dose of 8-OH-DPAT (150 micrograms/kg, i.v.) produced a 60% decrease in the height of the 300 mV oxidation peak in the median raphe, whereas only a 20% decrease was obtained in the dentate gyrus. In contrast, the non-selective 5-HT1 agonist RU 24,969 (10 mg/kg, i.p.) caused a 70% reduction of the 300 mV peak height in both the dorsal and median raphe and a 50% decrease in both the frontal cortex and the dentate gyrus. Moreover, although a high dose of 8-OH-DPAT (150 micrograms/kg, i.v.) given alone reduced by 20% the amplitude of the oxidative peak in the dentate gyrus, subsequent administration of RU 24,969 (10 mg/kg, i.p.) caused a further 30% diminution of the oxidative peak height. The greater responsiveness of dorsal as compared to median raphe 5-HT systems to 5-HT1A receptor agonists was confirmed in two further series of experiments. First, the microiontophoretic application of 8-OH-DPAT directly onto 5-HT neurons was three times more potent in suppressing the firing rate of dorsal raphe 5-HT neurons than that of their median raphe congeners. Second, 8-OH-DPAT and buspirone were ten and four times, respectively, more potent in decreasing 5-HT synthesis in the frontal cortex than in the hippocampus.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals with a portacaval shunt exhibit several biochemical abnormalities in plasma and brain similar to patients with portal-systemic encephalopathy, i.e., hyperammonemia, amino acid imbalance, and neurotransmitter disturbances. We investigated behavior and brain monoamine metabolism in operated, sham-operated, and nonoperated rats 1 day and 2, 4, and 6 weeks after operation. In order to quantitate the turnover in the brain indoleamine and catecholamine systems, 5-hydroxytryptophan (5-HTP) and dihydroxyphenylalanine were measured after decarboxylase inhibition with NSD 1015. The brains were dissected into five regions. All rats with the shunt had high plasma ammonia concentrations. Behavioral tests revealed a reduction in spontaneous locomotion 2, 4, and 6 weeks after portacaval shunt and reduced exploratory behavior compared with control rats. These changes coincided with profound alterations of the indoleaminergic system. As early as 1 day after surgery, rats with the shunt showed a marked increase in the accumulation of 5-HTP in all brain regions, indicating an enhanced tryptophan hydroxylase activity. The changes in indoleamine synthesis were most profound in the cortex and the midbrain. Only minor alterations of the catecholaminergic system could be detected. The alterations in behavior and indoleamine neurotransmitter metabolism may be pathophysiologically interrelated and may serve as the basis for experimental studies of portal-systemic encephalopathy.
Substantial concentrations of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA), comparable to those found in brain tissue, were measured in the small pial vessels of the rat, rabbit and cat. Both rat and rabbit pial vessels exhibited a high affinity uptake process with kinetic parameters similar to those identified for the cerebral cortex. Labelled 5-HT, taken up by isolated rabbit pial vessels was released, in a calcium-dependent manner, by potassium-induced depolarization. Various pharmacological manipulations were carried out in the rat. Systemic administration of the 5-HT precursor, 5-hydroxytryptophan and the monoamine oxidase inhibitor, pargyline, significantly increased the concentration of 5-HT in the pial vessels; in contrast, two depleting agents (p-chloroamphetamine and reserpine) and the tryptophan hydroxylase inhibitor, p-chlorophenylalanine, all decreased the perivascular 5-HT levels. A serotonergic antagonist (methysergide) and a 5-HT receptor agonist (MK 212) respectively increased and decreased the concentrations of 5-HIAA in the pial vessels. These pharmacologically induced changes observed in pial vessels were not dissimilar from those noted for cortical tissue. Electrolytic lesions of the nuclei raphes medianus and/or dorsalis markedly decreased the levels of 5-HT and 5-HIAA in these small cerebral arterioles. Electrical stimulation of these nuclei decreased 5-HT although 5-HIAA concentrations tended to increase. A number of conclusions may be drawn from these studies. Thus, there is a serotonergic innervation of the cerebral circulation in several laboratory species which unequivocally originates in the raphé nuclei. Furthermore, these perivascular fibres possess synthetic, storage, release, inactivation and autoregulatory processes for 5-HT which, when further elucidated, may offer some rationale for the treatment of those cerebrovascular diseases in which this neurotransmitter and vasoactive agent is believed to be of pathological importance.
The effect of gamma-aminobutyric acid (GABA) mimetics on extracellular concentrations of 5-hydroxyindoleacetic acid (5-HIAA) (as measured by differential pulse voltammetry with carbon fiber electrodes) in the dorsal raphé has been investigated in the rat. Systemic administration of dipropylacetamide decreased extracellular 5-HIAA to a similar extent, and within a comparable time course, in the dorsal raphé and striatum. Similar results were obtained after intradorsal raphé infusion of muscimol (100 ng). In contrast, local infusion of tetrodotoxin into the dorsal raphé failed to alter serotonin metabolism in this area. It is concluded that GABA depresses serotonin metabolism not only in nerve endings, but also in dendrites (and/or cell bodies) of serotonergic neurons.
Several putative neurotransmitter amino acids and monoamine metabolites were measured in the cerebrospinal fluid of spontaneously photosensitive baboons (Papio papio) at different periods with varying degrees of photosensitivity in the same animals. At maximum photosensitivity the inhibitory amino acids gamma-aminobutyric acid and taurine were lower, and those of asparagine (metabolite of the excitatory amino acid aspartate) were higher, than when the animals were not photosensitive. Thus a decreased inhibition and perhaps increased excitation correlates with the level of photosensitivity.
The potential role of the habenula in the transsynaptic regulation of the activity of ascending dopaminergic systems has been investigated in the rat by studying the effect of an acute interruption of impulse traffic in the diencephalic conduction system (stria medullaris-habenula-fasciculus retroflexus) and of pharmacological manipulation of various neurotransmitter systems in the interpeduncular nucleus on dopamine metabolism in several dopaminergic projection fields. The bilateral infusion of tetrodotoxin into the fasciculus retroflexus (which conveys the habenulointerpeduncular tract) of conscious rats markedly increased homovanillic acid levels and dopamine synthesis and utilization in the medial prefrontal cortex, nucleus accumbens, olfactory tubercle and striatum. Similar changes in dopamine metabolism were observed in these areas after bilateral infusion of tetrodotoxin into the stria medullaris (which conveys most of the afferents to the habenula). Infusion of atropine (0.4-1 micrograms) into the interpeduncular nucleus increased homovanillic acid concentrations and dopamine utilization in the medial prefrontal cortex and nucleus accumbens but not in the olfactory tubercle and striatum. Moreover, intra-interpeduncular injection of oxotremorine (17 micrograms) antagonized the increase in dopamine utilization in the nucleus accumbens (but not in the olfactory tubercle) induced by an intrafasciculus retroflexus infusion of tetrodotoxin. Local infusion of naloxone (20 micrograms) into the interpeduncular nucleus increased homovanillic acid concentrations in the nucleus accumbens and olfactory tubercle but not in the medial prefrontal cortex and striatum. In contrast, intra-interpeduncular nucleus infusion of the substance P antagonist D-Arg1, D-Pro2, D-Trp7,9, Leu11-substance P or of substance P antiserum failed to alter homovanillic acid levels in the 4 dopamine-rich areas investigated. Finally, intraraphé medianus (but not intraraphé dorsalis) infusion of muscimol (25 ng) moderately increased dopamine synthesis in the nucleus accumbens and striatum. The present findings suggest that the habenulointerpeduncular pathways exert a tonic inhibitory influence on mesocortical, mesolimbic and mesostriatal dopaminergic neurons. Cholinergic and/or opioid peptidergic neurons coursing through the fasciculus retroflexus as well as ascending serotonergic neurons originating in the raphé medianus could take part in this inhibitory control of ascending dopaminergic neurons.
To investigate the possible alterations of spinal cord monoaminergic pathways in Parkinson's disease, the levels of dopamine, homovanillic acid, noradrenaline, serotonin and 5-hydroxyindoleacetic acid have been measured in different subregions of the lumbar spinal cord in control subjects and parkinsonian patients. Substantial amounts of these compounds were found in the dorsal, intermediate and ventral grey matter portions and in the white matter of the spinal cord; the levels of serotonin and its metabolite being the highest. In parkinsonian patients, lumbar spinal cord dopamine and homovanillic acid levels were similar to those in the control subjects, whereas the concentrations of noradrenaline, serotonin and its metabolites were clearly subnormal in the different parts of the cord, the depletion of noradrenaline being the most pronounced. These data suggest that lumbar spinal cord noradrenergic and serotonergic, but not dopaminergic, systems are damaged in Parkinson's disease.
Rats were subjected to total hepatectomy or a sham operation and infused 5 h with 10% glucose solution. The metabolism of indoleamines and catecholamines was studied in five regions of the brain and two regions of the spinal cord by using a decarboxylase inhibitor (NSD 1015) blocking the conversion of 5-hydroxytryptophan (5-HTP) to serotonin and DOPA to dopamine. In the brain the concentrations of 5-HTP, serotonin, and 5-hydroxyindoleacetic acid (5-HIAA) were elevated in all regions compared with controls except for serotonin in the mesencephalon-pons. In the spinal cord the concentrations of 5-HIAA were elevated whereas the concentrations of 5-HTP and serotonin were unchanged. The concentrations of DOPA were increased in the mesencephalon-pons whereas those of norepinephrine were decreased in cortex and mesencephalon-pons compared with controls. The results suggest an increased synthesis rate of the indoleamines in the brain and probably also of the catecholamines in the mesencephalon-pons at 5 h after hepatectomy.
This report described a new microdissection procedure to evaluate the regional distribution of neuromediators in the rat spinal cord. Different segments are first divided into sagittal slices. From these, different grey and white matter regions can be microdissected. This permits selective biochemical measurements in different laminae, including the area around the central canal. White and grey matter can also be differentially analysed. Using HPLC with electrochemical detection, we report on the regional analysis of biogenic amines as well as uric acid. An increase in 5-hydroxytryptamine (5-HT) levels was observed from cervical to lumbar segments. 5-HT levels were highest in the motoneurone samples (lamina IX) of the lumbar cord. The next highest levels were found in lamina X, followed by the intermediate grey matter laminae and the dorsal horn. Small amounts of 5-HT were detected in the white matter. Interestingly, the ratio of 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT was greatest in the white matter and least in the motoneurone sample. Norepinephrine (NE) levels were higher in the thoracic than in the cervical or the lumbar cord. The highest levels were found in the lamina X in thoracic segments. With this exception, no marked laminar difference in NE levels was observed. Dopamine (DA) levels were highest in the dorsal horn of the cervical and the thoracic cord, the next highest levels were found in the intermediate grey matter and lamina X in the same segments. The distribution of uric acid (UA) was comparable to that of NE: UA levels were highest in the thoracic cord, but no marked laminar difference was observed. On the other hand, UA levels in white matter generally exceeded those in the grey matter. These data indicate the value of a sagittal regional microdissection of the spinal cord. The ability to separately analyse different laminae of the cord (as well as differentiating grey and white matter) should prove useful in future studies of experimentally evoked changes in neurotransmitters within functionally distinct regions of the spinal cord.
Lumbar punctures were performed on four occasions over a 5-day period (8:30 a.m. on days 1, 3, and 5; 2:30 p.m. on day 2) on 10 normal volunteers (five of each sex; mean age, 27.7 years) to assess, with repeated sampling, the day-to-day variation of selected CSF parameters. Two subjects abstained from the lumbar puncture on day 5 due to headache after the third puncture. Lumbar CSF was analyzed for concentrations of free and total gamma-aminobutyric acid (GABA), homocarnosine, homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), total protein, albumin, and immunoglobulin (Ig)G. No significant concentration differences were found between the afternoon and next morning samples. No differences were found in concentrations of free GABA, total GABA, homocarnosine, 5-HIAA, or albumin across the study. In contrast, HVA concentrations significantly increased by day 5, whereas total protein and IgG decreased during the study. The most likely explanation for these changes involves the known concentration gradients in the CSF column.
The effects of a specific GABA receptor agonist, progabide, have been examined on local cerebral glucose utilization through the use of the autoradiographic [14C]2-deoxyglucose technique in conscious rats. The intraperitoneal administration of progabide (80-320 mg.kg-1) resulted in a heterogeneous pattern of significantly reduced glucose utilization throughout the 50 discrete regions of the brain that were studied. Insignificant decreases in local cerebral glucose use were noted following the low dose of progabide (80 mg.kg-1). Following progabide (160 mg.kg-1), reductions of approximately 20% in glucose utilization were observed in most of the extrapyramidal system (substantia nigra, pars compacta and reticulata; globus pallidus; caudate nucleus), some thalamic nuclei (lateral geniculate body, anterior and ventrolateral thalamic nuclei), a number of areas related to the limbic system (cingulate cortex; amygdala; hypothalamus; lateral habenula; nucleus accumbens; dorsal hippocampus as well as the CA3 field) and the raphé nuclei. In contrast, layer IV of the neocortex, the cerebellum and cerebellar nuclei, displayed only minimal (10-15%) reductions in glucose utilization. At the highest dose (320 mg.kg-1) examined, progabide effected widespread though heterogeneously distributed decreases in glucose use. The overall pattern of decreased glucose use seen with progabide was different from that noted with previously studied GABA-mimetic drugs, such as muscimol, except for those changes observed in the extrapyramidal and sensory-motor areas for which similar dose-response relationships occurred. A significant correlation was observed between progabide-induced decreases in glucose utilization and in serotonin synthesis in a number of brain areas. The pattern of progabide-induced changes in integrated functional activity is compatible with its neurochemical spectrum and could indicate the loci at which its anticonvulsant, antidepressant and extrapyramidal activities are initiated.
The cellular localization of the rat brain neutral endopeptidase (NEP, EC 3.4.24.11) was investigated by quantitative autoradiography of the enzyme inhibitor ([3H]HACBO-Gly) after lesions of the striatum, nigrostriatal and corticostriatal pathways. The effect of these lesions on NEP levels was compared with that on δ and μ opioid receptors, selectively labeled with [3H]Tyr-d-Thr-Gly-Leu-Thr ([3H]DTLET) and [3H]Tyr-d-Ala-Gly-MePhe-Glycinol ([3H]DAGO), respectively. Twenty-one days after injection of kainate in the caudate putamen (CP), the NEP level was locally decreased (52%) but the time course of this decrease was different from that of μ and δ opioid receptors: [3H]DAGO binding was diminished by 40% from day 2 whereas that of [3H]DTLET was reduced by 51% from day 7. Kainic acid injection in the CP induced in the globus pallidus (GP) and substantia nigra (SN) a distant reduction of the 3 opioid markers. Likewise after injection of colchicine in the CP, [3H]HACBO-Gly binding was decreased in the GP (60%) and SN (58%), [3H]DTLET binding was reduced by 54 and 55% in the GP and SN, respectively and [3H]DAGO labeling was diminished by 49% in the GP, and 58% in the SN. Finally, lesion of the nigrostriatal dopamine pathway by 6-hydroxydopamine did not induce any change of NEP level in the CP and GP whereas δ and μ opioid receptor levels were diminished respectively by 25 and 29% in the CP, and 45 and 39% in the GP, a new fiding of the present study. Taken together these data suggest that NEP is in part associated with striatal intrinsic neurons. In the GP and SN, a large part of NEP seems to be presynaptically associated with nerve terminals endowed with μ and δ opioid receptors, which originate from efferent striatal neurons. In contrast to opioid receptors in the CP, the NEP appears not to be associated with dopaminergic nerve terminals originating from the SN. Cortical ablation did not affect any of the opioid markers.
The neuroanatomical site of the inhibitory influence of anxiolytics on central serotonergic transmission has been investigated in the rat by studying the effect of systemic or intracerebral administration of these drugs on cerebral serotonin (5-HT) synthesis. Systemic administration of diazepam (3 mg/kg s.c.) or flunitrazepam (1 mg/kg, s.c.) caused a reduction of 5-HT synthesis (as measured by the accumulation of 5-hydroxytryptophan after inhibition of aromatic amino acid decarboxylase) in the hippocampus but not in the cerebral cortex, striatum, cerebellum or spinal cord of the rat. Zopiclone (22 mg/kg, s.c.) decreased the amine synthesis in hippocampus, striatum and prefrontal cortex. The decrease of hippocampal 5-HT synthesis induced by diazepam (5 mg/kg, s.c.) was antagonized by the benzodiazepine antagonist Ro 15-1788 (2 X 30 mg/kg, s.c.) but not by bicuculline (2 X 1 mg/kg, s.c.). Acute cerebral hemitransection or electrolytic lesion of the fasciculus retroflexus did not prevent the ability of diazepam (5 mg/kg, i.p.) to diminish hippocampal 5-HT synthesis. Local infusion of diazepam (15 micrograms) of flurazepam (1.5 micrograms) into the hippocampus of conscious rats (via indwelling cannulae) markedly reduced 5-HT synthesis in this brain area whereas infusion of these drugs into the raphé medianus (origin of the serotonergic afferents to the hippocampus) failed to affect hippocampal 5-HT synthesis. In contrast, local injection of muscimol (25-150 ng) into the raphé medianus reduced 5-HT synthesis in the hippocampus. This effect of muscimol was potentiated by a systemic administration of diazepam or an intra-raphé medianus infusion of flurazepam (at doses or concentrations which exhibited no intrinsic activity). It is concluded from these data that anxiolytic drugs exert an inhibitory influence on hippocampal serotonergic neurons which is mediated primarily via GABA-independent benzodiazepine receptors located in the vicinity of serotonergic nerve terminals.
Utilizing reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection and optimization of the mobile phase using factorial designs and a constructed computer program to predict chromatograms, it has been possible to obtain a satisfactory resolution of seventeen of the major monoamine neurotransmitters, precursors and metabolites. A rapid (less than 25 min) isocratic system for the simultaneous determination of 3,4-dihydroxyphenylalanine, dopamine, dihydroxyphenylacetic acid, 3-methoxytyramine, homovanillic acid, norepinephrine, normetanephrine, 3,4-dihydroxyphenylethylene glycol, 3-methoxy-4-hydroxyphenylethylene glycol, epinephrine, metanephrine, vanillylmandelic acid, 5-hydroxytryptophan, serotonin, 5-hydroxytryptophol and 5-hydroxyindoleacetic acid in addition to the internal standard isoproterenol is presented. The optimization strategy included selection of variables to optimize by a reduced factorial design a detailed study of these variables by a complete factorial design, theoretical predictions of chromatograms by a constructed computer program and test on the HPLC system. This optimization strategy can easily be applied to any problem of solute separation by liquid chromatography.
D-1 and D-2 receptor densities, evaluated respectively by [3H]SCH 23390 and [3H]spiperone binding, and DARPP-32 (dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein-32K) concentrations, were studied in the brains of control and parkinsonian subjects postmortem. D-2 receptor density was unchanged in the putamen of parkinsonian patients. D-1 receptor density was unchanged in the putamen and substantia nigra pars reticulata (SNR) of parkinsonian patients, but decreased by 28% in the substantia nigra pars compacta (SNC). DARPP-32, which is localized in the same structures as D-1 receptors of which it is thought to represent the intracellular messenger, decreased by 45% in the putamen, 66% in the SNR, and 79% in the SNC. The decrease in D-1 receptors in the SNC may be due to degeneration of pallidonigral GABAergic neurons, but some of the D-1 receptors may be on the nigrostriatal dopaminergic neurons themselves. The dissociation between the alteration of D-1 receptor densities and DARPP-32 concentrations in both the striatum and substantia nigra, which are of the same order in the two structures, may be an index of functional hypoactivity of D-1 neurotransmission.
Using the technique of trans-striatal dialysis in halothane-anesthetized rats, we have studied the effects of intrastriatally infused N-methyl-D-aspartate (NMDA), kainate, and quisqualate on the liberation of endogenous striatal dopamine. The striatal infusion of NMDA (10(-3)-10(-2) M) or kainate (10(-4)-10(-2) M) but not of quisqualate (up to 10(-2) M) for one 20-min fraction provoked a dramatic increase in striatal dopamine efflux up to a maximum of 1,200 and 3,400% of basal levels for NMDA and kainate, respectively. NMDA (10(-3) M) evoked liberation of striatal dopamine was totally blocked by coinfusion of 2-amino-5-phosphonovalerate (2-APV; 5 X 10(-4) M) and by the systemic injection of phencyclidine (3 mg/kg i.p.). The effects of NMDA (10(-3) M) were also totally antagonized in a dose-dependent manner by the striatal coinfusion of atropine (10(-7)-10(-4) M), and abolished in rats that had received bilateral striatal ibotenate lesions (10 micrograms/1 microliter) 1 week prior to implantation of the dialysis fiber. The striatal infusion of tetrodotoxin (10(-6) M) reduced basal dopamine efflux by 60-70% and abolished the NMDA (10(-3) M)-evoked liberation of striatal dopamine. The effects of kainate (10(-3) M) on striatal dopamine efflux were only partially reduced by doses of 2-APV or atropine that totally blocked the NMDA response, and were also partially resistant to tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
Paroxetine is a selective and potent inhibitor of 5-hydroxytryptamine uptake into serotonergic neurons. The specific binding of [3H]paroxetine to rat cortical membranes at 22 degrees C was examined in this study. Our results indicate the presence of a single saturable high affinity binding component for [3H]paroxetine. Scatchard analysis revealed a Kd of 0.15 +/- 0.01 nM, and a Bmax of 549 +/- 36 fmol/mg protein. The kinetically derived dissociation constant was 0.034 +/- 0.008 nM. [3H]Paroxetine binding was inhibited selectively by 5-HT uptake blockers, and a good correlation was demonstrated between the potency of various drugs to inhibit [3H]paroxetine binding and [3H]5-hydroxytryptamine uptake. Also, lesions performed with the neurotoxin, 5,7-dihydroxytryptamine resulted in a 94% decrease in endogenous 5-hydroxytryptamine levels and concomitantly, a 90% reduction in [3H]paroxetine binding when compared to sham controls. These results indicate that the binding site labelled by [3H]paroxetine is associated with the neuronal 5-hydroxytryptamine transporter complex.
Acute injection of the gamma-aminobutyric acid (GABA) mimetics progabide, aminooxyacetic acid, gamma-acetylenic GABA and dipropylacetamide reduced 5-hydroxytryptophan (5-HTP) accumulation in serotonergic nerve terminal regions (prefrontal cortex, olfactory tubercle, septum, striatum, hypothalamus, hippocampus, substantia nigra, cerebellum and spinal cord) as well as in corresponding cell body areas (raphé dorsalis, medianus, pontis and magnus). This effect was antagonized by bicuculline. The inhibition of serotonin (5-HT) synthesis induced by a single progabide administration was accentuated on repeated treatment in the striatum, prefrontal cortex and cerebellum but was similar to that seen after acute treatment in the other areas. Local infusion of high concentrations of GABA or GABA mimetics into the striatum, septum or substantia nigra failed to modify 5-HTP accumulation in these areas. Cerebral hemitransection antagonized the ability of progabide (1200 mg/kg i.p.) to diminish 5-HTP accumulation in the striatum, hippocampus and prefrontal cortex. Intra-raphé dorsalis infusion of muscimol (0.1-100 ng) or GABA (1-100 micrograms) decreased 5-HT synthesis in the corresponding projection areas (e.g. striatum, substantia nigra, cortex) but not in the hippocampus or cerebellum. Conversely, intra-raphé medianus infusion of these drugs diminished 5-HTP accumulation in the corresponding projection areas (e.g. hippocampus, septum, cortex) but not in the striatum or cerebellum. Intra-raphé dorsalis or medianus injection of GABA antagonists (bicuculline, picrotoxinin, RU-5135) was without effect on cerebral 5-HT synthesis but antagonized the diminution of the amine synthesis observed in corresponding projection areas after intra-raphé dorsalis or medianus infusion of muscimol or GABA. These results suggest that GABA exerts an inhibitory (non-tonic) control over central serotonergic neurons which is mediated via GABA receptors located in the raphé nuclei.
In order to explore the anatomical nature of the inhibitory GABAergic control of cerebral serotonergic neurons exerted at the level of the anterior raphé cells in the rat, we have studied the effect of GABA agonist agents (given systemically or infused locally into the raphé dorsalis or medianus) on cerebral 5-hydroxytryptophan (5-HTP) accumulation after lesion or pharmacological manipulation of various raphé inputs. Destruction of noradrenergic pathways by local injection of 6-hydroxydopamine in the pedunculus cerebellaris superior or by systemic injection of DSP-4 (50 mg/kg i.p.), or alteration of central dopaminergic transmission (by systemic administration of apomorphine or haloperidol) failed to modify the ability of progabide (400 mg/kg i.p.) or dipropylacetamide (150 mg/kg i.p.) to diminish 5-HTP accumulation in the striatum, hippocampus and substantia nigra. In contrast, electrolytic lesion of the habenular nuclei blocked the ability of these compounds (given systemically) to reduce 5-HTP accumulation both in serotonergic nerve terminal and cell body (raphé dorsalis and medianus) areas. A similar blockade of the effects of GABA mimetics was seen after ibotenate-induced lesion of the habenula but not after electrolytic lesion of the stria medullaris (which conveys most of the afferents to the habenula). Acute cessation of impulse flow in the habenulo-raphé tract also prevented the depamide-induced diminution of cerebral 5-HTP accumulation. Finally, interruption of nerve transmission in the habenulo-raphé pathways (by means of electrolytic lesion of the habenula or fasciculus retroflexus) blocked the ability of GABA (100 micrograms) or muscimol (50 ng) injected into the raphé dorsalis or medianus to reduce 5-HTP accumulation in the corresponding serotonergic nerve terminal areas. It is concluded that the GABAergic inhibition of ascending serotonergic neurons exerted in the raphé dorsalis and medianus depends upon an ongoing neuronal activity in the habenulo-raphé pathways; GABA may exert its inhibitory control over serotonergic neurons by tuning down a facilitatory influence on these cells exerted by the habenula.
The effect of manipulations of noradrenergic neuronal activity on the levels of the deaminated metabolites of dopamine in the locus coeruleus has been investigated in the rat. Antidromic stimulation of the locus coeruleus increased the levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) in this area. Conversely, local infusion of tetrodotoxin into the locus coeruleus reduced the levels of these metabolites in this region. After systemic injection, idazoxan increased whereas clonidine diminished DOPAC, HVA and MOPEG levels in the locus coeruleus either in normal animals or in animals bearing a lesion of A9 and A10 dopaminergic cells. These results suggest that the formation of dopamine deaminated metabolites in noradrenergic cell bodies is dependent upon, and may serve as an index of, central noradrenergic neuronal activity.
In order to explore the nature of the facilitatory GABAergic control of cerebral noradrenergic neurons, we have studied the effect of a variety of GABA mimetics (given systemically or injected locally into brain areas containing noradrenergic cell bodies or terminals) on several indices of noradrenaline turnover in the rat brain. Systemic administration of both direct and indirect acting GABA mimetics enhanced; 1) the pargyline induced accumulation of normetanephrine in the hypothalamus; 2) total DOPEG levels in a number of brain regions innervated by noradrenergic neurons; 3) both DOPAC and MOPEG levels in noradrenergic cell body areas (A1, A2 and A6). These effects are probably mediated by GABAA receptors as specific GABAA or mixed GABAA/GABAB agonists but not the GABAB agonist baclofen enhanced noradrenaline turnover. Interruption of noradrenergic impulse flow (by local injection of tetrodotoxin or by hemitransection) blocked the ability of progabide to increase DOPEG concentrations in the hypothalamus and cerebral cortex. Similarly, the co-administration of clonidine with progabide antagonized the progabide-induced increase in hypothalamic total DOPEG levels. Co-administration of yohimbine with progabide provoked an additive effect on hypothalamic DOPEG levels at moderate but not at high doses of yohimbine. Thus, the acceleration of noradrenaline turnover induced by GABA mimetics appears to depend on ongoing activity in noradrenergic neurons and occurs via an increase in neuronal discharges. Local injection of muscimol into the nucleus accumbens or hypothalamus failed to affect DOPEG levels in these structures; similarly, local injection of muscimol into the locus coeruleus failed to modify DOPEG levels in corresponding noradrenergic projection areas. These data indicate that the GABAergic influence is not exerted via GABA receptors located on noradrenergic cell bodies or nerve endings. Furthermore, since systemically administered progabide still increased hypothalamic DOPEG levels after ibotenate-induced destruction of the hypothalamic neuronal cell bodies, a presynaptic modulation of noradrenergic neurons by local GABAergic interneurons is excluded. Chemical destruction of serotoninergic pathways or enhancement of 5-HT transmission by quipazine failed to alter the ability of progabide to increase cerebral DOPEG levels. Moreover, scopolamine or naloxone also failed to affect the progabide-induced increase in cerebral DOPEG levels.(ABSTRACT TRUNCATED AT 400 WORDS)
In slices of the rat hippocampus, alpha 2-adrenoceptors located presynaptically on serotonergic nerve terminals modulate the electrically evoked calcium-dependent release of [3H]serotonin [( 3H]5HT). We have investigated the effects of a naturally occurring trace amine, beta-phenylethylamine (beta-PEA), on noradrenergic transmission in the rat hippocampus. Under experimental conditions in which MAO of type B is inhibited by deprenyl-exposure to beta-PEA (0.1-10 microM) facilitates the spontaneous outflow of [3H]noradrenaline and inhibits the electrically evoked release of [3H]5HT. The inhibitory effect of beta-PEA (3 microM) on the evoked release of [3H]5HT was antagonized by the alpha 2-adrenoceptor antagonist idazoxan at 1 microM, and by pretreatment with alpha-methyl-p-tyrosine (alpha-MpT, 300 mg/kg i.p., 2 h). The inhibition of tyrosine hydroxylase activity by alpha-MpT does not modify the inhibition of the evoked release of [3H]5HT by the alpha 2-adrenoceptor agonist, 6-fluoronoradrenaline, or the serotonin receptor agonist, 5-methoxytryptamine. Pretreatment with the neurotoxin DSP4 (50 mg/kg i.p., 10 days) markedly antagonized the inhibitory action of beta-PEA on [3H]5HT release. These results indicate that the noradrenaline-releasing action of beta-PEA inhibits the electrically evoked release of [3H]5HT through the activation of alpha 2-adrenoceptors. This inhibitory effect appears to be mediated exclusively through the release of newly synthesized noradrenaline, and does not involve the direct activation by beta-PEA of the inhibitory 5HT autoreceptors which modulate [3H]5HT release in the rat hippocampus.
Gas chromatography-mass spectrometry was used to quantitate serotonin, N-acetylserotonin, 5-methoxytryptamine, and melatonin in single rat pineal glands. After gas chromatographic separation, the
ion density of specific fragments of each indole was measured with mass spectrometry. Sensitivity of this indole assay is
of the order of 10-12 to 10-13 mole. Routinely, specificity is based on gas chromatographic retention time and the recording of the ion density generated
by specific fragments. Absolute identification of the extracted indoles was based on multiple ion detection.
A quantitative method for the analysis of 5-hydroxytryptamine in biological material is described. The method is based on high performance liquid chromatography (HPLC) with electrochemical detection. A simple purification on a weakly acidic ion exchange resin prior to the analysis gives quite clean samples and permits concentration of diluted samples. The chromatographic separation is performed on a reverse phase column with organic modifier added to an aqueous eluent. With this analytical system 25 pg of 5-hydroxytryptamine can be detected.
A new method for the concurrent extraction and quantification of tryptophan (Trp), tryptamine (T), indole-3-acetic acid (IAA), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5-HIAA) in samples of rat brain is presented. Homogenization is carried out in 0.1 n HCl containing 1 n KCl and 0.2% NaHSO3. After centrifugation at 100,000g, the supernatant is percolated through a column of XAD-2 resin, eluted with distilled methanol, and the resulting eluate is evaporated to dryness. The dry residue is then derivatized to yield the pentafluoropropionated (PFP) and methylpentafluoropropionated (Me-PFP) derivatives. Identification and quantification is readily achieved by gas chromatography-mass fragmentographic analysis on a OV-17 or Dexsil 300 column. Endogenous levels in whole rat brain established by this method are IAA, 13,1 ± 2.0 ng/g (n = 6); T, less than 380 pg/g (n = 6); Trp, 4.16 ± 0.23 μg/g (n = 6); 5-HIAA, 442 ± 24 ng/g (n = 6); and 5-HT, 526 ± 81 ng/g (n = 5).
A new method for the concurrent assay of three tryptophan metabolites at the picomole level is described. The method has been developed for blood, urine, cerebrospinal fluid, and tissue samples such as whole brain, brain parts, and endocrine glands. Tryptophan itself, serotonin, and 5-hydroxyindoleacetic acid are isolated initially on extraction columns, eluted with a suitable solvent, and injected onto a liquid chromatograph with an amperometric detector. This general approach may be applicable to a variety of other tryptophan metabolites and should be useful in both research and clinical investigations.
An N-acetylserotonin/melatonin radioimmunoassay (NAS/Mel RIA) and a Mel RIA were developed to measure NAS and Mel contents in tissues of rats and chickens. Anti-NAS and anti-Mel sera were produced by immunization of rabbits with NAS-M-bovine serum albumin (BSA) and Mel-M-BSA, respectively. Anti-NAS serum used in the NAS/Mel RIA reacts equally well with NAS and Mel, while anti-Mel serum used in the Mel/RIA reacts specifically with Mel. The NAS and Mel levels in the pineal and Harderian gland, the retina and the brain of rats and chickens and Mel levels in chicken serum were determined using these RIAs. Levels of NAS and Mel in the rat pineal demonstrated diurnal rhythms with high levels during the dark period and low levels during the light period. The indole levels determined in this study correlate well with those obtained by other methods.
GABA was injected intraperitoneally to rats in single doses of 2.5 to 1500 mg/kg. Thirty minutes after injection a dose-dependent increase in dopamine (DA) and a decrease in noradrenaline (NA) content were observed in the brain. However, in the lowest dose range these levels showed small but significant changes in the opposite direction. The accumulation of dopa after inhibition of the aromatic L-aminoacid decarboxylase was enhanced by i.p. GABA both in DA- and in NA-predominated brain regions, the dose-response relations being complex. Increased levels of serotonin (5-HT), 5-hydroxyindole-acetic acid (5-HIAA) and tryptophan as well as enhanced accumulation of 5-hydroxytryptophan, induced by decarboxylase inhibition were also observed.
The general pattern of effects was similar to that previously observed after intracerebroventricular injection of GABA, although the intraperitoneal doses required were higher. It is suggested that a certain penetration of GABA from the blood into the brain can occur, leading to changes in the physiological activity of monoaminergic neurons.
—Rat cerebral slices were incubated in oxygenated Krebs-Ringer bicarbonate glucose saline, and the uptake of Li+ was measured after periods of 15 s to 5 min. Saturation was not seen within the concentrations of Li+ employed (0·5-2·0 mm). The half-time of the uptake was 7·9 min. At steady state, after 1 h incubation, the concentration of Li+ in the tissue was linearly related to that of the medium (0·5-1·5 mm Li+) with a concentration ratio of 1·29–1·66. The concentrations of K+ and Na+ in the slices incubated without Li+ were found to be (μmol/g incubated wt, mean ±s.d.) 63·8 ± 9·6 and 96·2 ± 7·8 respectively (n = 28). In the presence of media with 1·5 mm -Li+, the K+ and Na+ in the slices were 56·2 ± 8·8 and 101·0 ± 7·7 respectively (n = 37). The concentration of Li+ in the slices, after 1 h incubation, increased in a non linear way as the concentration of K+ in the medium was decreased within a range of 0·10 mm-K+. In the absence of K+ in the medium the uptake of Li+ was approx 50% higher than in the presence of 4·9 mm-K+. There was an inverse linear relationship between the concentration of Li+ in the slices and that of Ca2+ in the medium within the range of 0-5·2 mm (-0·13 mm-Li+/mm Ca2+). The concentration of Li+ in the slices increased by approx 10% when the Mg2+ in the medium was increased from 1·3 mm to 2·6 mm. Changes of the concentration of Na+ between 120 mm and 170 mm in the medium had no significant effect on the Li+ uptake.
An inexpensive pump is described for high pressure liquid chromatography which can be assembled from readily available parts. This pressurized tank type apparatus can be used with conventional liquid chromatography detectors, or with the spectrophotometers or fluorometers available in most laboratories. The system has been applied to measurement of specific amino acids in brain tissue: taurine, γ-aminobutyric acid and 5-hydroxytryptophan. The assays, which can measure less than 50 pmol, require only 3-7 min per sample, and require no sample preparation, other than precipitation of proteins. The apparatus can perform complex separations for analytical work, but its simplicity, high speed, and ease of sample preparation make it also suitable for enzymatic and clinical studies.
A method is described for the selective isolation of 5 HT from a CSF sample. The indoleamine is then converted into [ 3H]melatonin in conditions sightly different from those of Saavedra. Finally, TLC of the radioactive product is carried out. As little as 10 to 20 pg of 5 HT can be determined in 1-3 ml of CSF using this procedure.
A rapid and sensitive method for measuring 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, using o-phthalaldehyde and L-cysteine, is presented, enabling both compounds to be measured in small areas of rat brain.
: Tyrosine hydroxylase (TH, EC 1.14.16.2), glutamate decarboxylase (GAD, EC 4.1.1.15), and choline acetyltransferase (ChAT, EC 2.3.1.6), activities were used as markers of the presence of catecholaminergic, GABAergic, and cholinergic neurons in human brain. The disparity in the absolute values obtained in control caudate nucleus led us to study the pattern of distribution of these enzymes in given brain regions, with the idea that the pattern might be altered specifically in pathological conditions. The tridimensional distribution of TH, GAD, and ChAT activities was investigated within the caudate nucleus, the putarnen, and the pallidurn. In control patients, opposite rostro-caudal and rnedio-lateral gradients appeared for GAD and CAT; ChAT activity was higher at the caudal level and GAD activity was higher at the rostral level of caudate nucleus and putamen. In the medio-lateral extent of putarnen and pallidurn, ChAT activity was highest in the lateral part of the putarnen; GAD was highest in the medial segment of the pallidum. Only GAD presented a particular dorsoventral pattern, the enzyme activity being highest in the ventral part of the caudate nucleus and the putarnen. No reproducible distribution was observed for TH. In Parkinsonian patients, a decrease of TH and GAD activities with abolition of gradients was observed, whereas the ChAT activity and pattern were not modified.
Tryptophan and many of its indole metabolites were separated using reversed-phase high-performance liquid chromatography (HPLC) and determined using electrochemical detection. This was accomplished isocratically using an acetate--citric acid eluent with various amounts of methanol. Brain and pineal tissue was analyzed for several tryptophan metabolites. Tissue preparation required only homogenization in acidic solution and centrifugation prior to application to the HPLC column. Detection limits in the low picogram range were found for those indoles separated.
A rapid and sensitive method has been outlined for the measurement of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) utilizing a weak cation-exchange resin and liquid chromatography with electrochemical detection. The sensitivity of the procedure allows measurement of the amine in punches of rat substantia nigra even after local injection of the neurotoxin 5,7-dihydroxytryptamine. Increases in 5-HT and decreases in 5-HIAA concentrations after pargyline, and selective increases in 5-HIAA concentrations after probenecid were detected in selected brain regions (nucleus accumbens, anterior striatum, substantia nigra). Thus, this procedure is sensitive enough to estimate 5-HT turnover in discrete nuclei of the rat brain.
A highly selective and sensitive method for the measurement of serotonin and its metabolites in brain tissue has been developed, based on reverse-phase liquid chromatography. The method combines a clean-up step on small, gravity-fed isolation columns with a liquid chromatograph utilizing an on-line sample enrichment procedure. This procedure significantly increases the sensitivity available, allowing determination of picomolar concentrations. Application of this technology is made to the determination of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in cerebrospinal fluid and small regions of the mammalian brain. As little as 1 mg. of tissue can be studied with a RSD of 4.46% for 5-HT and 5.98% for 5-HIAA.
An extremely sensitive and simple method for simultaneously measuring both serotonin and 5-hydroxyindoleacetic acid (5-HIAA) has been developed using liquid chromatography (LC) and electrochemical detection. Assay conditions are described which resolve and measure as little as 22 picograms of serotonin and its deaminated metabolite in deproteinated brain samples.
The effect of tiapride on HVA and 5-HIAA levels in the CSF drawn at pneumoencephalography (PEG) was studied. Five consecutive 5 ml fractions of CSF were drawn from control and tiapride-treated subjects. In both groups, a linear increase in HVA concentrations was found between the first and subsequent fractions. On the contrary, no significant difference in 5-HIAA concentrations was found in sequential CSF samples. Tiapride increased the mean HVA concentrations and caused a steeper caudocranial gradient of this metabolite but failed to modify 5-HIAA concentrations. The results suggest that tiapride blocks dopamine (DA) receptors and increases DA synthesis.
A simple and sensitive method for the concurrent determination of the monoamine metabolites MHPG, DOPAC, HVA and 5HIAA in brain samples is described. After solvent extraction at acid pH, the metabolites are separated by HPLC on a C18 reversed phase column using phosphate buffers. Detection and quantification are achieved using fluorescence and electrochemical detection in series. The method is applied to control samples of divers areas of human and non-human primate brain, and the distribution of results agrees well with those obtained by existing methods. The concentrations found also agreed well with literature values, and, for 5HIAA and DOPAC, with results obtained on parallel samples analysed by fluorimetry and by GC. Results for HVA however are higher than those obtained by GC, but agree well with literature values obtained by fluorimetry and by GCMS.
: The rate of tryptophan hydroxylation in vivo was estimated in discrete rat brain nuclei by measuring L-5-hydroxytryptophan (5-HTP) accumulated after pharmacological blockade of L-5-hydroxytryptophan decarboxylase by NSD 1015, using a sensitive radioenzymatic microassay. Endogenous serotonin, a major contaminant in this assay, was quantitatively removed by cationexchange chromatography prior to analysis. In non-treated animals, endogenous 5-HTP could be detected in small but measurable amounts. Following NSD 1015, accumulation occurred linearly for at least 30 min. At this time the recorded figures were two to six times higher when compared to values obtained in the same discrete structure from non-treated animals. This allows an accurate estimation of the rate of tryptophan hydroxylation in vivo in small fragments of grossly dissected brain regions (e.g. cortex) as well as in discrete nuclei containing either serotoninergic (5-HT) cell bodies (brain stem raphe nuclei) or 5-HT-terminals (e.g. catecholaminergic group A l, A2, A6.,. etc). Parachlorophenylalanine drastically reduced the rate of tryptophan hydroxylation in vivo in both terminal regions and raphe nuclei, with similar figures, 3 h or 3 days after injection. Chloral hydrate anaesthesia was attended by a transient decrease which appeared delayed in the raphe nuclei. Finally, pargyline pretreatment led to an 80% decrease in the forebrain, while no significant change appeared in the raphe nuclei. Thus, as illustrated by these few pharmacological manipulations, this method allows the study of the regulation of tryptophan hydroxylation in vivo with an improved anatomical resolution. Investigations can be carried out in the various raphe nuclei and their corresponding terminals in discrete brain areas simultaneously.
A high-performance liquid chromatographic procedure is described for the determination of 5-hydroxytryptamine or serotonin (5-OH-TRA), tryptophan (TRP), and 5-hydroxytryptophan (5-OH-TRP). The method, which is based on the separation on a sulfonated fluorocarbon polymer coated on pellicular silica support and measurement of the native fluorescence, has a detection limit of about 1 pmol serotonin (as well as of 5-OH-TRP) and 2 pmol TRP. In addition, it has the added advantage of specific product identification and quantitation. The method has been applied to the assay of serotonin in rat brain tissue. The procedure, which has been designed for use with an automatic sampler, has an analysis time of approximately 3 min per sample (approximately 20 assays per h).
Tryptophan, serotonin, 5-hydroxyindoleacetic acid, and homovanillic acid were determined in rat brain by the direct injection of a centrifuged tissue homogenate into a liquid chromatographic-fluorometric/amperometric system. The above indoles, along with melatonin, were also determined in single rat pineal glands. The utility of the system in determining several additional catechols and idoles in brain was examined.
A simple, rapid and specific method for the determination of serotonin and catecholamines in brain is described. After tissue homogenisation, catecholamines are isolated by adsorption onto alumina and elution with perchloric acid. Serotonin is isolated by extraction into n-heptanol and back-extraction into acid. High-performance liquid chromatography of the acid extracts is performed with a C18 reversed-phase column and simple mobile phases. Detection is by the intrinsic fluorescence of the amines on excitation at 200 nm. Detection limits are 100 pg for norepinephrine, 300 pg for dopamine and 20 pg for serotonin. The results are found to correlate well with a catechol O-methyl transferase radioenzymatic assay for catecholamines and a ninhydrin derivatisation procedure for serotonin.