Automated brain dialysis of acetylcholine in freely moving rats: detection of basal acetylcholine.
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Acetylcholine↗
Biomedical subjects
B H Westerink
Publications and source records attributed to B H Westerink.
Dopamine re-uptake inhibitors show inconsistent effects on the in vivo release of dopamine as measured by intracerebral dialysis in the rat.
The effects of the dopamine (DA) re-uptake blockers amfonelic acid and GBR 12909 on the in vivo release of DA were investigated. DA release was measured by means of a fully automated brain dialysis system. GBR 12909 induced a 3-fold increase in the output of DA; amfonelic acid was without effect. These results indicate a dissociation between the behavioural effects of DA re-uptake blockers and the effects on DA release. A strong synergistic rise of DA release was observed when haloperidol was co-administered with amfonelic acid or GBR 12909. The potentiation of DA release was completely prevented by infusion of 1 microM tetrodotoxin.
3,4-Dihydroxyphenylacetic Acid↗
Simultaneous determination of the rates of synthesis and metabolism of dopamine in various areas of the rat brain: application to the effects of (+)-amphetamine.
A method is described in which two different dopamine turnover estimations are combined. The method is based on coadministration of a DOPA-decarboxylase inhibitor and a monoamine-oxidase inhibitor. This approach allows the simultaneous calculation of the biosynthesis rate of dopamine, as well as the formation rate of its main metabolite 3,4-dihydroxyphenylacetic acid. No mutual interference of the two turnover methods was observed. The method was applied in a dose-effect study of the influence of (+)-amphetamine on the synthesis and metabolism of dopamine.
3,4-Dihydroxyphenylacetic Acid↗
The use of tetrodotoxin for the characterization of drug-enhanced dopamine release in conscious rats studied by brain dialysis.
The effect of TTX (infused during brain dialysis of the striatum and nucleus accumbens) on the in vivo release of dopamine, 3,4-dihydroxyphenylacetic acid and homovanillic acid, was investigated. In addition it was studied whether the increase in the release of dopamine, induced by various pharmacological treatments, was sensitive to TTX infusion. The following drugs were studied: haloperidol, amphetamine, haloperidol co-administered with GBR 12909, morphine and MPP+. Dialysis was carried out in the striatum, except for morphine, which was studied in the nucleus accumbens. The infusion of TTX revealed three different types of pharmacologically enhanced dopamine release in conscious rats. First, action potential dependent dopamine release (exocytosis), which was observed in untreated animals as well as in animals treated with haloperidol, haloperidol + GBR 12909, and morphine. Second, action potential independent release (carrier-mediated) was established in the case of amphetamine. Third, action potential independent DA release, probably caused by neurotoxic reactions was observed during MPP+ infusion.
1-Methyl-4-phenylpyridinium↗
Measurement of acetylcholine release in freely moving rats by means of automated intracerebral dialysis.
The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC-assay system that comprises a cation exchanger, a post-column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 microM) of neostigmine resulted in gradually increasing amounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.
Acetylcholine↗
Failure to detect 6-hydroxydopamine in rat striatum after the dopamine releasing drugs dexamphetamine, methylamphetamine and MPTP.
Large doses of dexamphetamine, methylamphetamine and MPTP failed to induce the formation of 6-hydroxydopamine (6-OHDA) in rat striatum. The potent dopamine (DA) releasing actions of these drugs and of the MPTP metabolite MPP+ were shown by intracerebral dialysis of DA in conscious rats but 6-OHDA was not detectable in the perfusates. Neurotoxic effects of these DA releasing drugs are therefore probably not due, as previously suggested, to 6-OHDA formation and its subsequent uptake into nerve terminals.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
Brain dialysis in conscious rats reveals an instantaneous massive release of striatal dopamine in response to MPP+.
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1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
Circadian variation of catecholamine excretion in rats: correlation with locomotor activity and effects of drugs.
Rats housed in metabolic cages were used to study the circadian variation in the urinary excretion of free catecholamines. Small samples of urine (25-100 microliter) were analyzed for adrenaline, noradrenaline and dopamine by high pressure liquid chromatography (HPLC) and electrochemical detection. Various ways in which the values for excretion of catecholamines can be expressed (per min; per ml; per mmol creatinine; as ratio over dopamine) were calculated and discussed. Correction for excretion of creatinine resulted in the lowest variations coefficient among the experimental data. The correction for creatinine removed the circadian rhythm present in the output of noradrenaline (NA) and dopamine (DA). Adrenaline corrected for creatinine still displayed a pronounced circadian variation which was related to the overall locomotor activity of the animals (as recorded by photocells). Collection of 1 hr samples instead of 3 hr samples resulted in a worsening of the relationship between the excretion of adrenaline and locomotor activity. Finally, the possibility that the DA antagonist haloperidol, the DA agonist dipropyl-5,6-2-amino-6,7-dihydroxytetrahydronaphtalene (dipropyl-5,6 ADTN) and the alpha-antagonist phentolamine, could modify the excretion of free urinary catecholamines was investigated. Haloperidol and 5,6-dipropyl-ADTN did not change the output of the catecholamines, but phentolamine induced a strong increase in the excretion of NA. The latter observation suggest that at least part of the excretion of NA may originate from peripheral noradrenergic neurotransmission.
Aging↗
Effect of various centrally acting drugs on the efflux of dopamine metabolites from the rat brain.
The influence of chlorpromazine, haloperidol, morphine, chloral hydrate, gamma-butyrolactone, probenecid, kainic acid, oxotremorine, pargyline, yohimbine, (+)-amphetamine, and cocaine on the efflux rate of 3,4-dihydroxyphenylacetic acid (DOPAC) from four brain areas was studied. All drugs studied except pargyline and morphine had an effect on the transport of DOPAC and homovanillic acid (HVA) from the brain. Nine drugs inhibited the efflux of DOPAC and HVA, whereas (+)-amphetamine stimulated this transport. These data suggest that most centrally acting drugs can interfere with the elimination of 3,4-dihydroxyphenylethylamine (DA or dopamine) metabolites from the brain. These effects are heterogeneously distributed throughout the brain and are probably related to indirect nonspecific drug effects. This implies that drug-induced changes in DA metabolite concentrations, especially when these changes are slight to moderate, cannot directly be translated to changes in the production rate of these metabolites. By studying five control groups, we concluded that formation and transport of DOPAC are not synchronized in the various brain areas.
3,4-Dihydroxyphenylacetic Acid↗
Chronic use of intracerebral dialysis for the in vivo measurement of 3,4-dihydroxyphenylethylamine and its metabolite 3,4-dihydroxyphenylacetic acid.
The intracerebral dialysis technique was studied with a method in which the rat was directly connected to the HPLC equipment. The effect of three pharmacological treatments [perfusion of 60 mmol K+ or 5 X 10(-5) M (+)-amphetamine or subcutaneous injection of 2 mg/kg (+)-amphetamine] on the release of 3,4-dihydroxyphenylethylamine (dopamine) and 3,4-dihydroxyphenylacetic acid was followed over a period of 7 days. The marked rise of dopamine output seen after infusion of K+ had almost disappeared on day 3. Tissue reactions around the membrane presumably formed a barrier preventing K+ from reaching dopaminergic terminals. In contrast, the pronounced rise in dopamine level after amphetamine (infused as well as systemically administered) was still present (although diminished) 8 days after implantation. It is concluded that, with certain restrictions, brain dialysis of dopamine is still useful several days after implantation of the membrane.
3,4-Dihydroxyphenylacetic Acid↗
24 h excretion patterns of free, conjugated and methylated catecholamines in man.
A method based on high performance liquid chromatography with electrochemical detection is described for quantification of adrenaline, noradrenaline, dopamine (free and total), metanephrine, normetanephrine, and 3-methoxytyramine (free) in urine samples. Purification of the samples is achieved on disposable Bio-Rad ion-exchange columns in combination with small Sephadex G 10 columns. A clear circadian rhythm was found in the 24 h excretion pattern of free adrenaline and noradrenaline for 4 volunteers (city bus drivers during a working day). No rhythms were seen in the 24 h excretion pattern of free or conjugated dopamine, conjugated adrenaline or conjugated noradrenaline, or of the methylated free catecholamines. It is concluded that in order to obtain information about the neurotransmitter function of peripheral catecholamines it is sufficient to determine the non-conjugated compounds; values for the conjugated and methylated metabolites contribute no extra information.
Adult↗
On the origin of dopamine and its metabolite in predominantly noradrenergic innervated brain areas.
The origin of dopamine (DA) and its metabolite in predominantly noradrenergic brain areas was investigated in rats with a unilateral electrolytic lesion of the locus coeruleus. Using the unlesioned side as a control, levels of DA, noradrenaline (NA), and the DA metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were measured in lateral hippocampus, cerebellum, frontal cortex, occipital cortex, brainstem and striatum. Lesion of the locus coeruleus decreased brain NA levels in those regions innervated by this nucleus, but had no effect on levels of DA and DOPAC in any of the regions studied. It is concluded that even in regions of very low DA concentration, DA and DOPAC probably originate from dopaminergic neurons rather than via NA formation in noradrenergic neurons.
3,4-Dihydroxyphenylacetic Acid↗
A simple, sensitive, and economic assay for choline and acetylcholine using HPLC, an enzyme reactor, and an electrochemical detector.
A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G-10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine.
Acetylcholine↗
Certain dopamine agonists increase dopamine synthesis in the striatum of rat brain.
N,N-Dipropylamino-5,6-dihydroxytetrahydronaphthalene (DP-5,6-ADTN) induced a rapid and dose-related rise in dopamine (DA) levels in the striatum of rat brain. The increase of DA was less pronounced in the tuberculum olfactorium and there was no change in the frontal cortex. DP-5,6-ADTN and DP-6,7-ADTN also caused a rapid increase in striatal DA synthesis (evaluated from the rise of 3,4-dihydroxyphenylalanine after decarboxylase inhibition) which lasted about 20 min and was followed by a decrease. The increase in DA synthesis rate after the DP-ADTNs was restricted to the striatum, only a decrease was observed in mesolimbic areas and frontal cortex. Dose-effect curves showed that the ED 50% for the stimulation of DA synthesis was somewhat higher (approx. 0.2 mumol/kg) than the ED 50% for the decreasing effect (approx. 0.01 mumol/kg). The increase in striatal DA biosynthesis was not seen after the DA agonists piribedil, TL-99 or apomorphine. Pretreatment of rats with gamma-hydroxybutyrolactone or haloperidol prevented the DP-5,6-ADTN-induced increase in DA synthesis.
4-Butyrolactone↗
Formation and metabolism of dopamine in nine areas of the rat brain: modifications by haloperidol.
The rate of removal of 3,4-dihydroxyphenylacetic acid (DOPAC) in nine rat brain areas (striatum, nucleus accumbens, tuberculum olfactorium, hypothalamus, lateral hippocampus, occipital cortex, brain stem, cerebellum, and retina) was calculated from its exponential decline after monoamine oxidase inhibition by pargyline. The experiments were carried out with rats pretreated with either saline or haloperidol. It appeared that the efficiency with which DOPAC was removed from the brain (expressed by the fractional rate constant k) varied considerably throughout the brain. Haloperidol dramatically decreased the k values, and in addition these effects differed widely in the various brain areas. Similarly to DOPAC, haloperidol had a pronounced retarding effect on the efflux of homovanillic acid (HVA) from the brain. These findings strongly suggest that great care should be taken when drug-induced alterations in DOPAC and HVA concentrations are interpreted as changes in dopaminergic activity. The dopamine (DA) concentrations were measured in the same experiments, but it appeared that the pargyline-induced rise in DA was of limited use for the estimation of the synthesis rate of the amine. We calculated the rate of catecholamine synthesis in the nine brain areas from the rise of 3,4-dihydroxyphenylalanine (DOPA) during decarboxylase inhibition. In saline- as well as in haloperidol-pretreated rats it was found that the total catecholamine synthesis rate in the typical dopaminergic areas (striatum, nucleus accumbens, and tuberculum olfactorium) was of the same order of magnitude as the DOPAC rate of removal. This confirms that DOPAC formation is quantitatively the main route of degradation in these brain areas.(ABSTRACT TRUNCATED AT 250 WORDS)
3,4-Dihydroxyphenylacetic Acid↗
Determination of normetanephrine, 3,4-dihydroxyphenylethyleneglycol (free and total), and 3-methoxy-4-hydroxyphenylethyleneglycol (free and total) in rat brain by high-performance liquid chromatography with electrochemical detection and effects of drugs on regional concentrations.
A new, fast and sensitive assay for normetanephrine (NM), free and total 3,4-dihydroxyphenylethyleneglycol (DOPEG), and free and total 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG) in brain tissue is described. The method is based on high-performance liquid chromatography with electrochemical detection. Small Sephadex G 10 columns were used for prepurification. This permitted the additional isolation and quantification of tyrosine, 3,4-dihydroxyphenylalanine, noradrenaline, dopamine, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid. The compounds were determined in six brain areas (striatum, cortex, hippocampus, hypothalamus, brainstem, and cerebellum). Most DOPEG and MOPEG in rat brain was present in the conjugated form, except for the cerebellum, where about 80% of MOPEG was nonconjugated. No postmortem effects on MOPEG levels were observed; a slight increase in DOPEG in certain brain areas was found in microwave-killed rats. The effects of clonidine, yohimbine, N,N-dipropyl-5,6-ADTN, and chlorpromazine on the concentrations of the five noradrenaline (NA) metabolites were determined. Free and total DOPEG and MOPEG provide similar information on NA metabolism, whereas NM (after monoamine oxidase inhibition) reflects a different type of interaction of drugs with NA metabolism. The similarity in the pattern of drug-induced changes in NA metabolism in the various brain areas suggests that adrenoreceptors mediating NA metabolism are homogeneously distributed throughout the brain.
Animals↗
The release of endogenous dopamine and its metabolites from rat striatum as detected in push-pull perfusates: effects of systematically administered drugs.
The release of dopamine (DA) and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) was determined in push-pull perfusates from the striatum of the anaesthetized rat with high pressure liquid chromatography and electrochemical detection. Striking differences were found between the released amounts of DA (less than 7 fmol/min) and those of the metabolites (approx 0.5 pmol/min). It was calculated that 5% of the DA metabolites left the striatum via the push-pull cannula. The effects of systemic application of apomorphine, (+)-amphetamine, haloperidol or haloperidol in combination with amfonelic acid on the release of DA and its metabolites, were investigated. The release rate of DOPAC correlated well with the drug-induced changes of these metabolites in striatal tissue, however, this was not the case for HVA. The increase of striatal levels of DOPAC and HVA induced by the anaesthesia is a serious limitation of the method. To enable reliable estimation of the release of DA, 1 microM (+)-amphetamine had to be added to the perfusion medium (which may have modified the origin of DOPAC and HVA in the perfusates). (+)-Amphetamine (5 mg/kg, intravenous) induced a two- to threefold increase in the release of DA. Systemic administration of apomorphine, haloperidol or haloperidol in combination with amfonelic acid did not change the output of the neurotransmitter in the push-pull cannula. This implies that neuroleptics cause only a small and transient rise in DA release, whereas the pronounced and persisting increase in DOPAC and HVA seen after neuroleptics is the result of enhanced metabolism related to increased synthesis of DA.
3,4-Dihydroxyphenylacetic Acid↗
In vivo dopamine receptor binding studies with a non-radioactively labeled agonist, dipropyl-5,6-ADTN.
Evidence is presented that a low dose of peripherally administered N,N-dipropylamino-5,6-dihydroxytetralin (DiPr-5,6-ADTN) specifically labels dopamine (DA) receptors in rat brain. Concentrations of this potent DA receptor agonist were determined by a highly selective method using reversed phase liquid chromatography and amperometric detection. The binding characteristics satisfy all criteria regarding saturability, stereospecificity, regional distribution and relation with pharmacological effects that are associated with DA receptor interactions. A rough estimation of the density of binding sites in the striatum resulted in values of 60-70 pmol/g. Lesioning the nigrostriatal pathway does not significantly alter the amount of ligand bound, nor do pretreatments with serotonergic, alpha-adrenergic or beta-adrenergic antagonists. DiPr-5,6-ADTN has thus been shown to be a useful ligand for labeling central DA receptors and a powerful tool in the study of DA-ergic mechanisms.
3,4-Dihydroxyphenylacetic Acid↗