Margit Lindqvist

Evidence for a receptor-mediated feedback control of striatal tyrosine hydroxylase activity

WE have previously reported that cutting the nigrostriatal dopamine-carrying axons unexpectedly results in a transient increase in the rate of tyrosine hydroxylation in the rat forebrain (Carlsson, Kehr & others, 1972). It was suggested that striatal tyrosine hydroxylase activity is controlled via dopamine receptors at the synaptic cleft : when the impulse flow is interrupted by axotomy, the concentration of dopamine in the synaptic cleft decreases, and the ensuing reduction of dopamine receptor activity gives rise to a feedback activation of tyrosine hydroxylase, located in the striatal dopaminecarrying nerve terminals. The experiments now reported were made to test the above hypothesis. We argued that stimulation and blockade of dopamine receptors should result in inhibition and activation, respectively, of striatal tyrosine hydroxylase activity. Male Sprague-Dawley rats, 210-340 g, were used. Axotomy of the nigrostriatal dopamine fibres was performed under ether anaesthesia on the left side by means of a transverse cerebral hemisection, as previously described (BCdard, Carlsson&Lindqvist, 1972). At the same time (or in some experiments after 1 h) the aromatic amino-acid decarboxylase was inhibited by an intraperitoneal injection of NSD 101 5 (3-hydroxybenzylhydrazine HCl, 100 mg/kg). The animals were decapitated 30 min after the injection. The forebrains were analysed for dopa (Kehr, Carlsson & Lindqvist, 1972) and dopamine (Atack, to be published). For a direct activation of dopamine receptors, apomorphine HCl, 15 mg/kgf was injected intraperitoneally 7 min before the transection, and for blockade of these receptors haloperidol was given 1 h before the transection ( 5 mg/kg intraperitoneally, for references see AndCn, Carlsson & Haggendal, 1969). In some experiments both agents were given to the same animals. The levels of dopa in the forebrains are given in Fig. 1. As previously reported, inhibition of the aromatic amino acid decarboxylase causes the accumulation of dopa, which cannot be detected in the normal brain. This accumulation appears to be a useful indicator of the rate of tyrosine hydroxylation (Carlsson, Davis & others, to be published). In animals transected and treated simultaneously with NSD 101 5, the

Effect of ethanol on the hydroxylation of tyrosine and tryptophan in rat brain in vivo.

Various doses of ethanol were injected intraperitoneally into rats. After 20 min, 3‐hydroxybenzylhydrazine (NSD 1015), an inhibitor of aromatic amino‐acid decarboxylase was injected and 30 min later the animals were killed. The amount of dopa accumulating in brain as a consequence of decarboxylase inhibition was significantly increased by moderate doses of ethanol (up to 4 g kg−1). There was no corresponding effect on 5‐hydroxytryptophan. The effect on dopa was found both in a dopamine‐ and a noradrenaline‐dominated area of the brain, suggesting that catecholamine synthesis is stimulated by ethanol in both types of neuron. The data lend support to the hypothesis of Carlsson, Engel & Svensson (1972) that cerebral catecholamines are involved in the central stimulation induced by ethanol.

A method for the determination of 3,4-dihydroxyphenylalanine (DOPA) in brain

SummaryThe method is an extension of the chromatographic separation procedure for 5-HIAA and 5-HTP published by Lindqvist (1971). Brain tissue is extracted with perchloric acid. After adjustment of the pH to 2 the extract is passed through a Dowex 50W, X-4 column. 5-HIAA is eluted with 60% aqueous methanol, DOPA and tyrosine with 0.1 M sodium citrate buffer pH 4.5 and 5-HTP and tryptophan with 0.1 M sodium phosphate buffer pH 6.5. If the above procedure is combined with that published by Atack and Magnusson (1970) 9 different compounds can be estimated after a single column procedure. DOPA is assayed fluorimetrically using a modification of the trihydroxy-indole (THI) assay for noradrenaline (Bertler et al., 1958). The level of DOPA found in a normal rat brain was below the sensitivity of the method (10 ng/g). After inhibition of the aromatic amino acid decarboxylase a compound indistinguishable from authentic l-DOPA accumulated in the rat brain.

Conjoint native and orthophthaldialdehyde-condensate assays for the fluorimetric determination of 5-hydroxyindoles in brain

SummaryProcedures for the fluorimetric assay of 5-hydroxytryptamine, 5-hydroxytryptophan and 5-hydroxyindole-3-acetic acid in tissues are described. The assay procedures are based both on the native fluorescence of 5-hydroxyindoles in strong acid and on the considerably higher fluorescence intensity of the fluorophores obtained by means of condensation with orthophthaldialdehyde. A tissue blank procedure, utilizing potassium ferricyanide and cysteine, common to both assays in the joint procedure, is incorporated. The assays have been adjusted to previously described chromatographic procedures, using a strongly acidic cation exchange column, in which these 5-hydroxyindoles and their precursor, tryptophan, and also other biogenic amines and related compounds, are isolated from a single Dowex 50 column. Data illustrating the reproducibility, sensitivity and increased specificity of the overall procedures compare favourably with previously published results. The possibility of the differential estimation of different 5-hydroxyindoles in a mixture, by means of simultaneous determinations of native and orthophthaldialdehyde-condensate fluorescence in the joint assays, is pointed out.

Central and peripheral monoaminergic membrane-pump blockade by some addictive analgesics and antihistamines

The ability of various analgesics and antihistamines to block the amine‐uptake mechanism (the so‐named membrane pump) of central and peripheral monoamine‐storing neurons was investigated in mice. Activities indicating such blockade were observed within both groups but did not seem to correlate with analgesic or antihistaminic activity. The antihistamine chlorpheniramine proved remarkably potent on central 5‐hydroxytryptamine neurons.

Postmortal accumulation of 3-methoxytyramine in brain

SummaryRats were decapitated and the brains stored in situ at +37° C. A rapid accumulation of 3-methoxytyramine occurred during the first hour. The accumulation was enhanced by pargyline pretreatment. It was less rapid at lower temperatures.The postmortal loss of dopamine corresponded to the formation of methoxytyramine in the pargyline-treated rats but exceeded this formation in non-treated rats.Large amounts of methoxytyramine were also recovered from the basal ganglia of two human brains.

Catecholamine synthesis in rat brain after axotomy: Interaction between apomorphine and haloperidol

SummaryAxotomy of the ascending monoaminergic fibers by means of a complete cerebral hemitransection stimulated the formation of dopa during 30 min after inhibition of the aromatic amino acid decarboxylase with 3-hydroxybenzylhydrazine HCl, 100 mg/kg i.p., in c. striatum and the dopamine-rich part of the limbic system. Apomorphine, 0.5 mg/kg i.p., antagonized the accumulation of dopa not only on the intact but also on the lesioned side. Haloperidol, 2 mg/kg i.p., stimulated dopa accumulation on the intact side but could not further stimulate the increase in dopa caused by transection. When both drugs were given together, the inhibitory effect of apomorphine was fully counteracted by haloperidol on both sides. In the predominantly noradrenaline-innervated occipito-temporal cortex dopa formation was slightly higher on the lesioned than on the intact side and was not markedly influenced by apomorphine.In the rest of the hemispheres the apomorphine-induced decrease in dopa formation was more pronounced on the intact than on the lesioned side and was fully antagonized by haloperidol.The dopamine concentration was slightly higher in the lesioned c. striatum as compared to the intact side irrespective of the drugs administered. In c. striatum and the limbic system haloperidol caused a decrease in dopamine on the intact side which was not antagonized by additional treatment with apomorphine.Hemitransection caused a decrease in noradrenaline especially in the hemisphere portion. Neither apomorphine nor haloperidol or both drugs in combination changed the latter effect.In general, the tyrosine concentration tended to be higher on the lesioned than on the intact side in all brain structures investigated.The data support the view that a local receptor-mediated feedback mechanism exists which is controlling dopamine synthesis even in the absence of impulse flow.

Metatyrosine as a tool for selective protection of catecholamine stores against reserpine

Abstract Metatyrosine, injected i.p. to mice in single or repeated doses of 100 to 400 mg/kg, caused a marked, though short-lasting depletion of brain noradrenaline (NA) and dopamine (DA) and of heart NA, whereas brain 5-hydroxytryptamine (5-HT) was essentially unaffected. When given in repeated doses shortly before and after the injection of reserpine (3 mg/kg i.p.), metatyrosine caused partial but significant protection against the catecholamine-depleting action of reserpine in the brain, whereas brain 5-HT and heart NA were not, or at most slightly, protected. These effects on monoamine levels were observed 24 hr after the injection of reserpine. The protective activity of metatyrosine on brain NA was absent in animals pretreated with protriptyline (repeated doses of 10 mg/kg i.p.), whereas brain DA remained protected. Metatyrosine, with or without protriptyline pretreatment, markedly prevented the gross syndrome induced by reserpine. The hypothermic action of reserpine was only partially prevented. It is concluded that depletion of brain catecholamines, notably DA, plays an important role in the gross behavioural action of reserpine.

Effect of acute transection on the synthesis and turnover of 5-HT in the rat spinal cord

SummaryThe acute interruption of impulse flow in the descendent spinal 5-HT fibre systems by means of a spinal transection caused an approximately 50% decrease in 5-hydroxyindoleacetic acid levels below the lesion, indicating a reduced 5-HT turnover. Likewise, the accumulation of 5-hydroxytryptophan after inhibition of the aromatic amino acid decarboxylase was reduced by about 50% below the lesion, indicating a reduced rate of tryptophan hydroxylation. Both changes could still be seen after saturation of tryptophan hydroxylase by tryptophan loading. This immediate decrease in tryptophan hydroxylase activity after transection of the spinal cord probably took place without any loss of the total amount of enzyme, and may indicate an influence of nerve impulses on a factor modulating the activity of the enzyme. Three days after transection a pronounced decrease in tryptophan hydroxylation rate and in 5-HT level was observed below the lesion, probably due to degeneration of 5-HT fibre systems; above the lesion the 5-HIAA level was elevated.

Accumulation of 5‐hydroxytryptophan in mouse brain after decarboxylase inhibition

The amount of 5-hydroxytryptophan (5-HTP) normally present in the brain is too small to be detected by any of the methods so far available. Wiegand & Scherfling (1962) have reported a content of 5-HTP in mouse brain of less than 0.1 pg/g and Lindqvist (unpublished) has found a value of less than 0.07 pg/g rat brain and less than 0.03 pg/g mouse brain. We have now investigated the accumulation of 5-HTP in mouse brain after decarboxylase inhibition. Groups of 13-14 white female mice (NMRI), 18-24 g, were injected with a single dose of the aromatic amino-acid decarboxylase inhibitor Ro 4-4602[N1-(~~-seryl)-N2(2,3,4-trihydroxybenzyl)hydrazine], 800 mg/kg intraperitoneally. The animals were decapitated at time intervals after the injection, the brains quickly dissected and each brain immediately homogenized with ice-cold perchloric acid containing ascorbic acid and EDTA (disodium ethylenediamine tetra-acetate). The interval between killing an animal and homogenization of the brain was less than 15 s. 5-HTP was isolated on a Dowex 50, X-4 column according to an unpublished method of Lindqvist.