S. Bourgoin

The topographical distribution of serotoninergic terminals in the spinal cord of the cat: biochemical mapping by the combined use of microdissection and microassay procedures.

The topographical distribution of 5-HT in the spinal cord of the cat was established by the combined use of a microdissection technique and an enzymic microassay for the indoleamine. The procedure consisted of determining the 5-HT content in microdiscs of tissue (0.07 cu.mm) punched out in various zones of frontal sections of the spinal cord at the level of the cervical and lumbar enlargements. In both cases, the concentration of 5-HT in the grey matter was at least twice as high as that in the white matter. The motoneuron area of the ventral horn contained the highest level of 5-HT (402–472 pg) in both enlargements. In the dorsal horn 5-HT was particularly concentrated in the lateral part of the substantia gelatinosa, where its level (∼-320pg) reached about 75 % that found in the motoneuron area. A close value for 5-HT concentration (337–357 pg) was measured in the central area bordering the ependymal canal. A thoracic transection (Th9–10) induced a progressive decay in 5-HT concentrations below the lesion without altering that found in cervical enlargement. A time-course study has shown that 5-HT was almost undetectable in the lumbar enlargement on the 21st day after the thoracic transection. The selective electrolytic lesion of the nucleus raphe dorsalis did not alter the levels and distribution of 5-HT in the lumbar enlargement. In contrast, a partial lesion of the nucleus raphe magnus (40%) induced a significant decrease in 5-HT levels in this region. This effect was particularly pronounced in the dorsal horns where the 5-HT levels in the substantia gelatinosa were reduced by 44%.

Biochemical evidence for the 5-HT agonist properties of PAT (8-hydroxy-2-(di-n-propylamino)tetralin) in the rat brain

In vitro investigations revealed that PAT (8-hydroxy-2-(n-dipropylamino)tetralin) interacted with postsynaptic 5-HT receptors in the rat brain: the drug stimulated 5-HT-sensitive adenylate cyclase in homogenates of colliculi from new-born rats (KAapp 8.6 microM) and inhibited the specific binding of [3H]5-HT to 5-HT1 sites. The PAT-induced inhibition of [3H]5-HT binding showed marked regional differences compatible with a preferential interaction of PAT (IC50 2 nM) with the 5-HT1A subclass. As previously seen with 5-HT agonists, the efficacy of PAT for displacing [3H]5-HT bound to hippocampal membranes was markedly increased by Mn2+ (1 mM) and reduced by GTP (0.1 mM). PAT also affected presynaptic 5-HT metabolism since it inhibited competitively (Ki 1.4 microM) [3H]5-HT uptake into cortical synaptosomes and reduced (in the presence of the 5-HT uptake inhibitor fluoxetine) the K+-evoked release of [3H]5-HT previously taken up or newly synthesized from [3H]tryptophan in cortical or striatal slices. This latter effect was prevented by 5-HT antagonists (methiothepin, metergoline) suggesting that it was mediated by the stimulation of presynaptic 5-HT autoreceptors by PAT. Like 5-HT, PAT counteracted the stimulatory effect of K+-induced depolarization on the synthesis of [3H]5-HT from [3H]tryptophan in cortical slices. It is concluded that PAT is a potent 5-HT agonist acting on both post- and presynaptic 5-HT receptors in the rat brain.

The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat

The topographical distribution of serotoninergic terminals in the neostriatum of the rat and the caudate nucleus of the cat was established owing to the combined use of microdissection techniques and biochemical microassays. The density of 5-HT terminals in various areas of both structures was quantified first by measuring 5-HT levels in microdiscs of frozen tissue. Since the high affinity uptake process for 5-HT appeared undamaged in isotonic homogenates of previously frozen (--5 degrees C) tissues, it was possible to confirm the findings obtained with the measurement of 5-HT levels by also determining 5-HT uptake activity in these microdiscs. However, in the rat neostriatum, but not in the cat caudate nucleus, [3H]5-HT even at a very low extracellular concentration (4.4 -x 10(-8) M) was taken up not only by serotoninergic terminals but also to a significant extent by dopaminergic terminals. In presence of benztropine, this second component was suppressed and [3H]5-HT uptake activity could then be considered as a specific marker of serotoninergic terminals also in the neostriatum of the rat. In both species, 5-HT terminals were mainly localized in the ventrocaudal area of the structure. In this area, 5-HT levels were among the highest values found in the brain (17 ng/mg protein). The density of 5-HT terminals decreased progressively from the acudal to the rostral planes of the neostriatum in rats or the caudate nucleus in cats. The poorest area, i.e. the dorsorostral zone, contained about 4 times less 5-HT than the ventrocaudal zone of the structure. Electrolytic lesion of the dorsalis (B7) and centralis superior (B8) raphe nuclei during early life resulted in a large decrease of 5-HT levels (--90%) in various parts of the neostriatum of adult rats. The present findings might be of interest to further analyze the role of serotoninergic neurons in extrapyramidal functions.

The effects of quipazine on 5-HT metabolism in the rat brain

SummarySince quipazine is a potent 5-HT agonist in peripheral organs, its possible stimulatory effects on serotoninergic receptors in the rat brain were investigated. Quipazine administration (10 mg/kg, i.p.) induced a significant decrease in the synthesis and turnover rates of serotonin in the brain stem as well as in the forebrain. It is not likely that these changes were mediated by a negative feed-back mechanism triggered by adirect action of quipazine on central 5-HT postsynaptic receptors. Indeed, in contrast to LSD and 5-methoxy-N,N-dimethyltryptamine, this compound failed to activate the 5-HT sensitive adenylate cyclase in colliculi homogenates of newborn rats. However, quipazine exerted direct effects on serotoninergic terminals. It inhibited competitively the reuptake process in synaptosomes (Ki =1.38×10−7 M) and stimulated the K+ evoked release of newly synthesized3H-5-HT in slices of the brain stem. Injected in vivo in a dose which affected 5-HT uptake and release, quipazine did not modify MAO activity. However, this activity was noncompetitively inhibited by high concentration of the drug in vitro (Ki=3.0×10−5 M). These actions are very likelyindirectly responsible for the stimulation of central 5-HT receptors.

In vitro and in vivo effects of kelatorphan on enkephalin metabolism in rodent brain

Biologically relevant assays were used to compare the potency of kelatorphan (N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]-L-alanine) as inhibitor of the peptidase-induced metabolism of enkephalins to that of bestatin, a non-specific inhibitor of aminopeptidase and thiorphan, a highly potent blocker of the neutral endopeptidase (EC 3.4.24.11) designated as enkephalinase. Kelatorphan almost completely inhibited the formation of the three metabolites [3H]Tyr, [3H]Tyr-Gly and [3H]Tyr-Gly-Gly produced by incubation of [3H][Tyr1,Met5]enkephalin with rat striatal slices. Co-administered with [Met5]enkephalin in mouse brain, kelatorphan was able to prevent by 80% the degradation of the exogenous peptide. Moreover, a mixture of thiorphan (1 microM) and bestatin (20 microM) or kelatorphan alone (20 microM) induced a 2.2 to 2.5-fold increase in endogenous [Met5]enkephalin overflow after evoked depolarization of superfused rat striatal slices. In this assay, kelatorphan was the only compound to increase by 63% the basal level of released [Met5]enkephalin. Kelatorphan was about 100 times less potent than bestatin to inhibit the total rat striatal aminopeptidases, but as efficient (IC50 = 4 X 10(-7) M) as bestatin to inhibit a minor aminopeptidase activity resembling aminopeptidase M. Therefore the reported enhanced analgesic potency of kelatorphan with regard to the association of bestatin and thiorphan is very likely related to its ability to almost completely inhibit enkephalin-degrading enzymes (including the Tyr-Gly releasing peptidase) and to its better selectivity for the biologically relevant aminopeptidase M. Kelatorphan would be a valuable probe, preferable to the association of bestatin and thiorphan, to investigate the physiological functions regulated by a phasic enkephalinergic activity.

Basic and Regulatory Mechanisms of In Vitro Release of Met‐Enkephalin from the Dorsal Zone of the Rat Spinal Cord

Abstract: Under control conditions, superfused slices of the dorsal half of the lumbar enlargement from adult rats released Met‐enkephalin‐like material (MELM) that behaved as authentic Met‐enkephalin under two different chromatographic procedures (Bio‐gel filtration, HPLC). MELM release increased markedly on exposure of slices to batrachotoxin (0.5 μM) or to an excess of K+ (28 and 56 mM instead of 5.6 mM). The K + ‐evoked release was totally dependent on the presence of Ca2+ in the super‐fusing fluid whereas the spontaneous efflux of MELM was only partially Ca2+‐dependent. Further experiments performed with tissues of polyarthritic rats indicated that the increase in their MELM levels was associated with a lower fractional rate constant of MELM release, therefore suggesting that spinal Met‐enkephalin turnover might be reduced in chronically suffering animals. Examination of the possible modulation of MELM release by various neuroactive compounds present within the dorsal horn revealed that cholecystokinin (10 μM), but not its desulphated derivative, substance P‐sulphoxide (10 μM), and to a lesser extent substance P, enhanced the K+‐evoked MELM release. In contrast, γ‐aminobutyric acid (10 μM) and (–)‐baclofen (1 μM) partially prevented the stimulatory effect of K+ on MELM release. Other compounds such as serotonin, somatostatin, and neurotensin altered neither the spontaneous nor the K+‐evoked release of MELM.

In vivo release of 5-HT in the lateral ventricle of the rat: effects of 5-hydroxytryptophan and tryptophan

The in vivo release of 5-HT was examined in the rat brain. For this purpose, the left lateral ventricle was perfused at a constant rate with an artificial CSF for several hours in animals anaesthetized with halothane. 5-HT was estimated in serial 1-h collected fractions. The amine was first isolated by adsorption on a Sephadex G-10 column and then assayed using the radioenzymatic method of Saavedra et al.37, slightly modified to improve its sensitivity. The quantity of 5-HT released spontaneously during the first hour fraction was 296 pg, it was lower (99 pg/h) in the following fractions. 5-HT released into the CSF may in great part originate from serotoninergic terminals localized in structures surrounding the ventricle. This was suggested by experiments in which exogenous [3H]5-HT or [3H]tryptophan were perfused through the lateral ventricle during a few hours. [3H]5-HT taken up or synthetized was mainly localized in structures surrounding the ventricular space. The acute injection of 5-hydroxytryptophan (100 mg/kg) induced an immediate important and long lasting increase of 5-HT release. In contrast the acute injection of tryptophan (100 mg/kg) led to a transient and moderate elevation of 5-HT release which was only detected during the second hour of perfusion. Curiously a similar pattern of transmitter release was observed following the constant intravenous infusion of the amino acid (70 mg/kg/h) except that the increase in 5-HT release was much more pronounced during the second hour than after the acute injection. Parallel experiments were made to determine the time course of the changes of free and total tryptophan levels in plasma and of those of tryptophan, 5-HT, and 5-hydroxyindoleacetic-acid (5-HIAA) in brain tissues, induced by the acute and long term administrations of tryptophan. Moreover the rate of 5-HT synthesis was estimated using the monoamine oxidase inhibition method 2 and 5 h after both tryptophan treatments in halothane anaesthetized rats. 5-HT levels and the synthesis rate of the transmitter were increased at 2 h (when both tryptophan treatments stimulated 5-HT release). Despite the presence of high tryptophan levels in plasma and tissues and of high 5-HT and 5-HIAA levels in tissues, the synthesis rate of 5-HT (as the 5-HT release) was similar to that of controls 5 h after the onset of tryptophan infusion. These results suggest that some relationships occurred between the changes in 5-HT SYNTHESIs and release after the first hour of perfusion. The absence of effects of tryptophan treatments on 5-HT release during the first hour of perfusion are also discussed.

Effects of LSD on synthesis and release of 5-HT in rat brain slices

Abstract The effects of LSD on 5-hydroxytryptamine (5-HT) metabolism were studied in rat hippocampal and striatal slices. One hour after in vivo treatment with the drug (1 mg/kg i.p.) the synthesis of [3H]5-HT from l -[3H]tryptophan ([3]Try) was only decreased in the striatum. This effect was associated with a significant reduction in the accumulation of [3H]Try in tissues, whereas endogenous levels of Try in the striatum and hippocampus were significantly increased by this treatment. Such modifications of 5-HT metabolism could not be reproduced by adding LSD directly into the incubating medium of control slices. The release of [3H]5-HT, synthetized from [3H]Try or taken up by tissues, was very much increased when concentrations of K+ in the incubating medium reached 30 or 50 m M. This effect was counteracted by LSD, the drug being administered previously in vivo or added in vitro. Finally, high concentrations of LSD (0.1 m M) added in vitro induced a reserpine-like effect on newly synthetized as well as exogenous [3H]5-HT: the amine was markedly inactivated in its acidic metabolite.

FEEDBACK REGULATION OF 5-HT SYNTHESIS IN RAT STRIATAL SLICES

The effects of changes in intraneuronal levels of 5‐HT induced by monoamine oxidase inhibitors (MAOI) given in vivo or exogenous 5‐HT added in vitro on 5‐HT synthesis in striatal slices of the rat have been examined. The synthesis of 5‐HT was estimated by the measurement of the total formation of [3H]5‐HT and [3H]5‐hydroxyindole acetic acid from [3H]tryptophan and by calculation of the conversion index (CI) of tryptophan into 5‐HT. The small formation of [3H]tryptamine and [3H]indole acetic acid from [3H]tryptophan was taken into account in the estimation of 5‐HT synthesis. Both MAOI pretreament (180 min) and 5‐HT (2·8 μM) inhibited synthesis. The latter effect persisted in catecholamine depleted tissues and was related to intraneuronal changes in 5‐HT levels, since it could be prevented by chlorimipramine. The inhibition of 5‐HT synthesis was related to the decreased conversion of tryptophan into 5‐hydroxytryptophan and could not be prevented by p‐chloro‐phenylalanine pretreatment which depleted 5‐HT levels or by dibutyryl cyclic AMP which normally stimulated 5‐HT synthesis. Tryptophan uptake in slices was not affected by exogenous 5‐HT. The various mechanisms possibly involved in the end product regulation process of 5‐HT synthesis are discussed.

PLASMA TRYPTOPHAN AND 5-HT METABOLISM IN THE CNS OF THE NEWBORN RAT

—The relationships between plasma tryptophan and 5‐HT metabolism in the CNS were studied in newborn rats and compared with adults. Both the concentration of free tryptophan in plasma and that of the amino‐acid in brain were much higher immediately after birth than later on. Drugs such as salicylate and chlordiazepoxide, which increased brain tryptophan concentrations in adults by displacing the plasma amino acid bound to serum albumin, were ineffective in newborn rats: most of the amino acid being already free in their plasma. The study of 5‐HT metabolism in brain stem slices revealed that the affinity of the uptake process for tryptophan was higher in newborn than in adult animals, whereas the reverse situation was observed for the enzyme complex involved in 5‐HT synthesis (lower apparent Km in adults). In addition, the catabolism of newly synthesized 5‐HT was more rapid in newborn than in adult tissues. Finally, the free state of tryptophan in plasma of newborn animals induced in brain both a high amino acid concentration and, in contrast to the situation observed in adults, a synthesis rate of 5‐HT very near its maximal value.