F. Hery

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 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.

Increased synthesis and utilization of serotonin in the central nervous system of the rat during paradoxical sleep deprivation

Summary The effects of a 96 h paradoxical sleep deprivation (PS) on the metabolism of serotonin have been studied in the brain stem-mesencephalon, cortex-diencephalon and spinal cord of rats. A marked increased formation of [ 3 H]serotonin was observed in tissues of PS-deprived animals after intracisternal administration of [ 3 H]tryptophan or after incubation with [ 3 H]tryptophan when compared with control animals. No effect could be seen when [ 3 H]5-hydroxytryptophan was used instead of [ 3 H]tryptophan in experiments in vivo as well as in vitro . As indicated by the changes on [ 3 H]tryptophan accumulation in tissues and on serotonin-specific activity in the experiment in vitro , the increased [ 3 H]serotonin synthesis in PS-deprived animals seemed to result from both an increased transport of [ 3 H]tryptophan and an increased rate of conversion of [ 3 H]tryptophan to [ 3 H]5-hydroxytryptophan. The utilization of brain [ 3 H]serotonin endogenously synthesized from [ 3 H]tryptophan was accelerated during PS deprivation. The increased turnover of serotonin in PS-deprived animals appeared to be related to the impossibility of triggering PS.

Control of the release of newly synthetized 3H-5-hydroxytryptamine by nicotinic and muscarinic receptors in rat hypothalamic slices

SummaryThe effects of various cholinergic agonists and antagonists on the spontaneous release of newly synthetized 3H-5-HT were examined in rat hypothalamic slices. 3H-5-HT was measured in incubating medium at the end of a 30 min incubation carried out with l-3H-tryptophan in the presence of the various durgs tested. ACh (10−5 M) in the presence of eserine (2×10−4 M), and carbachol (10−5 M) stimulated the release of 3H-5-HT. In contrast, oxotremorine (10−5 M) reduced the 3H-amine release. The effect of carbachol was blocked by two nicotinic blockers, mecamylamine (10−6 M) and d-tubocurarine (10−6 M). It was not reduced by the muscarinic antagonists, atropine (10−6 M) and scopolamine (10−6 M). In fact, each of two antagonists added alone to the incubating medium enhanced 3H-5-HT release. The scopolamine (10−6 M) stimulating effect on 3H-5-HT release was suppressed by d-tubocurarine (10−6 M). Finally, the inhibiting effect of oxotremorine on 3H-5-HT release was not prevented by d-tubocurarine (10−6 M) but was in the presence of atropine (10−6 M) or scopolamine (10−6 M).In the concentrations used in the release study, the cholinergic agonists and antagonists had no effect on the total formation of 3H-5-HT and 3H-5-HIAA from l-3H-tryptophan and on the accumulation of l-3H-tryptophan in tissues. In these concentrations, except for eserine, they did not affect the uptake of exogenous 3H-5-HT in hypothalamic synaptosomes (P2 fraction).These results suggest that cholinergic receptors of the muscarinic and nicotinic type are involved in the control of 3H-5-HT release; since the stimulation of the muscarinic and nicotonic cholinergic receptors resulted in an inhibition and an activation of 3H-5-HT release, respectively. As in the case of peripheral noradrenergic and central dopaminergic neurons the cholinergic receptors could be localized on serotoninergic terminals.

Daily variations of various parameters of serotonin metabolism in the rat brain. II. Circadian variations in serum and cerebral tryptophan levels: Lack of correlation with 5-HT turnover

Rats submitted to regular 12 h cycles of light and darkness for three weeks were sacrificed at various times of the day. 5-HT, 5-HIAA and tryptophan levels were estimated in the fronto-parietal cerebral cortex. Tyrosine and free and total tryptophan levels in serum were estimated in parallel. Significant circadian variations in 5-HT and 5-HIAA levels were found in cerebral tissues. The peaks of 5-HIAA levels were dectected during the lignt and dark periods respectively, the maximal fluctuations being seen between 17.00 h and 21.00 h, two times separating the light off. Important significant circadian variations in free and total serum tryptophan levels were also observed. In both cases, the maximal levels were found during the middle of the dark phase after the peak of 5-HIAA levels. The circadian rhythm of tyrosine levels in serum was in opposite phase with that of tryptophan (free or total). The diurnal changes in tryptophan content in cerebral tissues seemed thus related to those found in serum. Taking in consideration results obtained in previous studies 16,17 carried out in similar experimental conditions, it was concluded that the parallel increase in serum free tryptophan and in tissues 5-HIAA levels seen during the night were not related to a stimulation of 5-HT turnover. Indeed 5-HT synthesis is minimal at this time16.

5‐HYDROXYTRYPTAMINE CATABOLISM IN THE RAT BRAIN DURING ONTOGENESIS

Although the serotoninergic innervation is immature in the brains of young rats, the 5‐HIAA content is similar to that found in adults. As indicated by the ratio of 5‐HIAA to 5‐HT levels in the brain stem and the forebrain, the catabolism of the indolamine was more rapid during the first 3 postnatal weeks than in adults. This was contirmed by measuring the total formation of [3H]5‐HIAA from [3H]5‐HT newly synthesized from L‐[3H]tryptophan in brain stem slices of young and adult rats.

Rat brain stem tryptophan hydroxylase: mechanism of activation by calcium.

Abstract— The activity of soluble tryptophan hydroxylase from rat brain stem was increased in presence of mm concentrations of calcium. Similarly to that observed by treating the enzyme with sodium dodecyl sulphate or trypsin, this activation resulted mainly from an increased affinity of tryptophan hydroxylase for both its substrate, tryptophan, and the cofactor 2‐amino‐4‐hydroxy‐6‐methyl‐5,6,7,8‐tetrahydropteridine (6‐MPH4). In addition, the optimal pH for the enzymic activity was shifted from 7.6 to 7.9 following activation by calcium, sodium dodecyl sulphate or trypsin.

Phospholipid-induced activation of tryptophan hydroxylase from the rat brainstem

Abstract Phospholipids (phosphatidylserine, phosphatidylethanolamine and lysophosphatidylcholine, but not phosphatidylinositol) and gangliosides stimulated the activity of tryptophan hydroxylase extracted from the brainstem of adult rats. This effect was pH dependent; it was much more pronounced at alkaline pH (8.3) than at pH 7.6 (pH for tryptophan hydroxylase assay under standard conditions); this resulted in a shift of the optimal pH for tryptophan hydroxylase activity from 7.6 to 7.9 when phosphatidylserine was included in the assay mixture. Another change concerned the apparent affinity of the enzyme for its cofactor, 6-MPH 4 , which doubled in the presence of phosphatidylserine. These alterations did not completely resemble those induced by sodium dodecylsulfate (SDS), suggesting that the detergent properties of phosphatidylserine were not solely responsible for tryptophan hydroxylase activation. This conclusion was strengthened by the observation that phosphatidylserine and SDS exerted opposite effects on tryptophan hydroxylase from young (16 day-old) rats: at pH 7.6, SDS stimulated, whereas the phospholipid largely inhibited the enzymic activity. Pretreatment of the 35,000 g supernatant of an homogenate of brainstem from adult rats with phospholipase A or C resulted in the activation of tryptophan hydroxylase. This suggests that changes in the composition of endogenous phospholipids (notably the formation of lysophosphatides and the removal of possible inhibitory phospholipids) in the vicinity of tryptophan hydroxylase may alter its activity. These results are discussed in relation to the possible role of phospholipids in the control of tryptophan hydroxylase activity under physiological conditions.

Characteristics of tryptophan binding in the serum of the newborn rat.

During the very first period of postnatal life, tryptophan is almost entirely free in the serum of rats. This situation is in sharp contrast with the well-known ability of serum albumin to bind the essential amino acid in the adult. Three main factors accounted for the relative lack of binding during the early postnatal life when compared to the adult: (1) the lower concentration of serum albumin, the binding protein; (2) the inhibition of binding by nonesterified fatty acids, which were at a high level in the serum of young rats until weaning, and (3) the decreased number of available binding sites for tryptophan on the defatted serum albimin, whereas the apparent association constant of tryptophan binding to serum albumin was similar in newborn and adult. Since immunological characterization of newborn and adult serum albumins did not reveal a specific fetal serum albumin, we suggest that discrete changes at the association site for tryptophan are sufficient to induce large alteration in the binding capacity of the protein. In contrast to the situation observed in adult rats, serotonin synthesis in the brain of newborn animals is therefore not dependent on the equilibrium between bound and free tryptophan in serum.