F. Artaud

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.

l-Glutamate-evoked release of dopamine from synaptosomes of the rat striatum: Involvement of AMPA and N-methyl-d-aspartate receptors

Previously, using purified synaptosomes from the rat striatum, we have shown that agonists of D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors stimulate the release of [3H]dopamine continuously synthesized from [3H]tyrosine. Similar results were obtained with N-methyl-D-aspartate in the absence of magnesium. In the present study, using the same approach, attempts were made to determine whether in the presence of magnesium, the combined stimulation of AMPA receptors allows us to demonstrate the presynaptic facilitation of [3H]dopamine release through N-methyl-D-aspartate receptors. L-Glutamate (10(-3) M) markedly stimulated the release of [3H]dopamine from synaptosomes, this effect being about twice that found with AMPA (10(-3) M) while N-methyl-D-aspartate (10(-3) M) even in the presence of glycine (10(-6) M) was ineffective. In agreement with previous results, a stimulatory effect of N-methyl-D-aspartate and glycine was only observed in the absence of magnesium. This response was blocked by 6,7-dinitro-quinoxaline-2,3-dione (3 x 10(-5) M), confirming that this compound, generally used as an AMPA antagonist, also blocks N-methyl-D-aspartate receptors. The AMPA (10(-3) M)-evoked release of [3H]dopamine was markedly potentiated by the combined application of N-methyl-D-aspartate (10(-3) M) and glycine (10(-6) M) in the presence of strychnine, indicating that the concomitant activation of AMPA receptors removes the voltage-dependent magnesium block of N-methyl-D-aspartate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P and neurokinin A regulate by different mechanisms dopamine release from dendrites and nerve terminals of the nigrostriatal dopaminergic neurons

Numerous striatal neurons innervating the substantia nigra contain substance P and/or neurokinin A. In contrast to substance P or neurokinin A, little neurokinin B is found in the substantia nigra. This led us to compare the effects of nigral application of these tachykinins on the release of dopamine from dendrites and nerve terminals of nigrostriatal dopaminergic neurons. Experiments were made in halothane-anesthetized cats implanted with one push-pull cannula in the substantia nigra and another in the ipsilateral caudate nucleus [3H]Tyrosine was delivered continuously to each push-pull cannula and the release of newly synthesized [3H]dopamine measured in the superfusate. Unlike substance P or neurokinin A, neurokinin B (10(-8) M) applied for 30 min into the pars compacta of the substantia nigra was without effect on the release of [3H]dopamine from nerve terminals or dendrites. When either substance P (10(-8) M) or neurokinin A (10(-8) M) was applied into the pars compacta, the release of [3H]dopamine from nerve terminals was enhanced. While neurokinin A also stimulated the dendritic release of [3H]dopamine, this was reduced by substance P. At a lower concentration (10(-9) M), neurokinin A induced similar effects to those observed at 10(-8) M whereas substance P (10(-9) M) stimulated moderately [3H]dopamine release from nerve terminals but did not affect the dendritic release of the [3H]amine. When superfused into the pars reticulata, substance P (10(-8) M) still stimulated [3H]dopamine release from nerve terminals but not from dendrites while neurokinin A (10(-8) M) was without effect either in the caudate nucleus or the substantia nigra. Additional experiments were made to determine whether or not substance P (10(-8) M) or neurokinin A (10(-8) M) act directly on nigral dopaminergic neurons when applied into the pars compacta. The effects of substance P on [3H]dopamine release from nerve terminals and dendrites were prevented when 2-amino-6-trifluoromethoxy benzothiazole (10(-5) M), an antagonist of glutamatergic transmission, was applied continuously into the caudate nucleus. In contrast, the stimulatory effects of neurokinin A on [3H]dopamine release from nerve terminals and dendrites were insensitive to 2-amino-6-trifluoromethoxy benzothiazole (10(-5) M). These results suggest that neurokinin A, but not substance P, acts directly on dopaminergic cells. In the light of previous observations, we propose that the effects of substance P on dopaminergic transmission are mediated by a nigro-thalamo-cortico-striatal loop.(ABSTRACT TRUNCATED AT 400 WORDS)

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 met-enkephalin in the cat brain

Abstract Push-pull cannulae were implanted into the globus pallidus (anterior part), the caudate nucleus (medio-dorsal part) and the substantia nigra of halothane-anaesthesized cats to study the characteristics of the in vivo release of met-enkephalin. Met-enkephalin was measured by radioimmunoassay (sensitivity: 0.1 pg) and identified by Bio-Gel P 2 chromatography and high-pressure liquid chromatography. Under resting conditions, met-enkephalin was detected in perfusates from these three regions; in the globus pallidus, the rate of spontaneous release of met-enkephalin was 5–6 times higher than that observed in the caudate nucleus and the substantia nigra. The local application of either an excess (60 mM) of K + or of glutamate (10 μM) produced a marked increase in the rate of met-enkephalin release both from the globus pallidus and the caudate nucleus. Further analyses in the globus pallidus indicated that the depolarizing agents, veratridine (50 μM) and batrachotoxin (0.1 μM), produced a large increment in the rate of met-enkephalin release; this effect was completely prevented by the local application of tetrodotoxin (2 μM). The chemical (with 60 mM K + or 10 μM glutamate), electrical or mechanical stimulation of the dorsomedial part of the caudate nucleus failed to significantly alter the rate of met-enkephalin release in the ipsilateral globus pallidus, in spite of the high sensitivity of enkephalinergic nerve terminals in the globus pallidus itself to local stimuli. This observation argues against the existence of a major caudatopallidal enkephalinergic pathway.

Spontaneous and evoked release of methionine-enkephalin-like material from the rat spinal cord in vivo.

In vivo perfusion of the subarachnoid space with an artificial cerebrospinal fluid (CSF) in paralyzed halothane-anesthetized rats allowed the collection of methionine-enkephalin (Met-Enk)-like material (MELM) released from the spinal cord. Bio-Gel P2 chromatography and high-performance liquid chromatography showed that 65% of this material corresponded to authentic Met-Enk. Under resting conditions, about 1 pg of MELM per minute was regularly released for at least 3 h; for Met-Enk, this value corresponded to a fractional rate constant of 0.002% (i.e. tissue content of the pentapeptide which was released per minute from the whole spinal cord). Perfusion with K+-enriched (40-60 mM) CSF resulted in a marked enhancement (+ 150-200%) of spinal MELM release. Similarly, calibrated pinches of the muzzle and i.p. administration of acetic acid, two strong noxious stimuli in awake animals, induced a significant increase (+ 75-150%) in spinal MELM release. In contrast, pinches applied to the tail did not enhance but instead slightly reduced (-35%) MELM release from the rat spinal cord. These data suggest that mechanisms other than segmental controls could be involved in the activation of spinal enkephalinergic neurons by some nociceptive stimuli.

THE ROLE OF INTRANEURONAL 5‐HT AND OF TRYPTOPHAN HYDROXYLASE ACTIVATION IN THE CONTROL OF 5‐HT SYNTHESIS IN RAT BRAIN SLICES INCUBATED IN K+‐ENRICHED MEDIUM

Abstract— The incubation of brain stem slices from adult rats in a K+‐enriched medium containing a 5‐HT uptake inhibitor (fluoxetine) significantly increased their capacity to synthesize 5‐HT from tryptophan. The K+‐induced stimulation of 5‐HT synthesis was at least partly dependent on the depletion of the indoleamine in tissues since: (1) a good correlation was found between the respective changes in 5‐HT release and synthesis evoked by high K+ concentrations in the presence of various 5‐HT uptake inhibitors; (2) the modifications in endogenous 5‐HT levels produced by in vim treatments with drugs (reserpine, pargyline) or by incubating slices with 5‐HT altered the stimulating effect of high K+ concentrations and fluoxetine on 5‐HT synthesis; (3) the replacement of Ca2+ by Co2+ (4 mM) or EGTA (0.1 mM) in the incubating medium completely prevented the increased 5‐HT release and synthesis evoked by high K+ concentrations and fluoxetine.

Specific role of n-acetyl-aspartyl-glutamate in the in vivo regulation of dopamine release from dendrites and nerve terminals of nigrostriatal dopaminergic neurons in the cat

Levels of N-acetyl-aspartyl-glutamate measured by high-pressure liquid chromatography were found to be very high in the cat substantia nigra, particularly in the pars compacta, while those in the caudate nucleus were much lower. In halothane-anaesthetized cats implanted with push-pull cannulae, N-acetyl-aspartyl-glutamate (10(-8) M) induced a marked and prolonged release of newly synthesized [3H]dopamine, when infused into the posterior but not into the anterior part of the caudate nucleus. In contrast, in the presence of tetrodotoxin (10(-6) M), N-acetyl-aspartyl-glutamate (10(-8) M) reduced the residual release of [3H]dopamine; this effect was also more pronounced in the posterior than in the anterior part. In the conditions used, as indicated by experiments with [3H]N-acetyl-aspartyl-glutamate no glutamate was formed from the infused N-acetyl-aspartyl-glutamate. Ibotenate (10(-5) M) induced changes in [3H]dopamine release in both the absence and presence of tetrodotoxin, which were closely similar to those observed with N-acetyl-aspartyl-glutamate. Responses induced by either N-acetyl-aspartyl-glutamate or ibotenate were not mediated by N-methyl-D-aspartate receptors since N-methyl-D-aspartate stimulated the release of [3H]dopamine only when used in a high concentration (10(-4) M) and applied in a magnesium-free superfusion medium in both the presence of glycine (10(-6) M) and strychnine (10(-6) M). In addition, the stimulatory effect of N-methyl-D-aspartate persisted in the presence of tetrodotoxin; it was of similar amplitude in both parts of the caudate nucleus and of shorter duration than that evoked by either N-acetyl-aspartyl-glutamate or ibotenate alone. N-Acetyl-aspartyl-glutamate interacted with dopaminergic neurons not only presynaptically in the caudate nucleus but also in the substantia nigra since a marked increase in [3H]dopamine release was observed both from local dendrites and from nerve terminals in the ipsilateral caudate nucleus when N-acetyl-aspartyl-glutamate (10(-7) M) was infused locally into the substantia nigra pars compacta. No effect could be seen in contralateral structures. The isomer of natural N-acetyl-aspartyl-glutamate, beta-N-acetyl-aspartyl-glutamate (10(-7) M), had no effect on [3H]dopamine release when applied similarly in the substantia nigra, thus confirming the specificity of the action of N-acetyl-aspartyl-glutamate.

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.