Guido Maura

Cholinergic terminals in rat hippocampus possess 5-HT1B receptors mediating inhibition of acetylcholine release

The effects of 5-hydroxytryptamine (5-HT) on the release of [3H]acetylcholine ([3H]ACh) from rat hippocampal nerve endings were investigated using synaptosomes labelled with [3H]choline and depolarized in superfusion with 15 mM KCl. The release of [3H]ACh was concentration dependently inhibited by exogenous 5-HT. The concentration-response curve of 5-HT was shifted to the right in a parallel way by methiothepin. The 5-HT2 antagonists ketanserin or methysergide did not antagonize the effect of 5-HT. The 5-HT1 agonist 5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole (RU 24969) mimicked 5-HT, whereas the 5-HT1A selective agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was ineffective. When used as a 5-HT1A/5-HT1B antagonist, (-)propranolol antagonized 5-HT whereas spiperone (a 5-HT1A displacer) did not. The 5-HT1C selective antagonist mesulergine was also ineffective towards 5-HT. It can be concluded that hippocampal cholinergic terminals are endowed with inhibitory 5-HT receptors which appear to belong to the 5-HT1B subtype.

Serotonin autoreceptor in rat hippocampus: pharmacological characterization as a subtype of the 5-HT1 receptor

SummaryThe 5-hydroxytryptamine (5-HT) autoreceptors mediating inhibition of [3H]5-HT release in rat hippocampus have been characterized pharmacologically in terms of 5-HT receptor subtype by using superfused synaptosomes depolarized with 15 mM KCl. Exogenous 5-HT inhibited in a concentration-dependent way (pEC30=8.74) the K+-evoked release of [3H]5-HT. Methiothepin shifted the concentration-response curve of 5-HT to the right (pA2=8.62). The 5-HT2 receptor antagonists, ketanserin, methysergide or spiperone were ineffective against 5-HT. The 5-HT1 receptor agonist, 5-methoxy-3-[1,2,3,6-tetra-hydropyridin-4-yl]-1H-indole (RU 24969) mimicked 5-HT and was equipotent as an inhibitor of the release of [3H]5-HT. In contrast, the putative 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was almost ineffective at 1 μM. Finally, (−)propranolol, used as a non-selective 5-HT1A/5-HT1B receptor antagonist, shifted to the right (pA2=7.91) the concentration-response curve of 5-HT whereas the 5-HT1C receptor antagonist mesulergine was ineffective. In conclusion, 5-HT nerve terminals of rat hippocampus possess autoreceptors which appear to belong to the 5-HT1B subtype.

5-Hydroxytryptamine3 receptors sited on cholinergic axon terminals of human cerebral cortex mediate inhibition of acetylcholine release.

Abstract: Synaptosomes prepared from freshly obtained human cerebral cortex and labeled with [3H]choline have been used to investigate the modulation of [3H]acetylcholine ([3H]ACh) release by 5‐hydroxytryptamine (5‐HT). The Ca2+‐dependent release of [3H]‐ACh occurring when synaptosomes were exposed in supervision to 15 mM KCl was inhibited by 5‐HT (0.01‐1 μM) in a concentration‐dependent manner. The effect of 5‐HT was mimicked by 1‐phenylbiguanide, a 5‐HT3 receptor agonist, but not by 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, a 5‐HT1A receptor agonist. The 5‐HT3 receptor antagonists tropisetron and ondansetron blocked the effect of 5‐HT, whereas spiperone and ketanserin were ineffective. It is suggested that cholinergic axon terminals in the human cerebral cortex possess 5‐HT receptors that mediate inhibition of ACh release and appear to belong to the 5‐HT3 type.

Noradrenaline inhibits central serotonin release through alpha2-adrenoceptors located on serotonergic nerve terminals

SummaryThe effect of noradrenaline on the depolarization-evoked release of 3H-5-hydroxytryptamine was investigated in superfused synaptosomes prepared from rat cortex and hippocampus and prelabelled with the radioactive indoleamine. Noradrenaline reduced in a concentration-dependent way the release of 3H-5-hydroxytryptamine elicited by 15 mM KCl. The inhibition was counteracted by the alpha-adrenoceptor antagonists phentolamine or yohimbine, but not by prazosin. The results indicate that, in rat brain, the inhibition of 5-hydroxytryptamine release by noradrenaline is mediated by adrenoceptors of the alpha2-type localized on the terminal serotonergic fibres.

Presynaptic muscarinic receptors increase striatal dopamine release evoked by ‘quasi-physiological’ depolarization

The effects of acetylcholine on the release of [3H]dopamine was studied in superfused rat striatal synaptosomes prelabeled with the radioactive amine. The results confirm the presence of muscarinic presynaptic receptors mediating potentiation of the spontaneous release of the catecholamine. However, under depolarizing conditions, the release of dopamine evoked by 15 mM KC1 was increased by the activation of muscarinic receptors and not decreased, as previously found in striatal synaptosomes or slices depolarized with higher (50-60 mM) KC1 concentrations.

Acetylcholine release from rat hippocampal slices is modulated by 5-hydroxytryptamine

Experiments were performed with slices of rat hippocampus in order to investigate whether the release of acetylcholine in this area is modulated through 5-hydroxytryptamine (5-HT) receptors. The slices were prelabeled with [3H]choline then stimulated electrically twice for 4 min each at a frequency of 3 Hz. The overflow of tritium evoked was inhibited by exogenous 5-HT in a concentration-dependent manner. The 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HC1 ((+/-)-DOI), did not mimic 5-HT. The effect of 5-HT was antagonized by methiothepin but not by the 5-HT2 antagonist, ketanserin. The 5-HT1 agonist, 5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole (RU 24969), inhibited the electrically evoked overflow of tritium, whereas the 5-HT1A-selective agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), was ineffective. Methiothepin itself, but not ketanserin, increased the evoked overflow of tritium. In contrast, the overflow was inhibited by the 5-HT uptake blocker, 6-nitroquipazine. The evoked overflow was also reduced by d-fenfluramine, a serotonin releaser. The concentration-inhibition curve for d-fenfluramine was shifted to the right by methiothepin. It is concluded that the release of ACh in rat hippocampus may be tonically inhibited by 5-HT through the activation of receptors, possibly belonging to the 5-HT1B subtype.

Serotonin-glutamate interaction in rat cerebellum: involvement of 5-HT1 and 5-HT2 receptors

The effects of serotonin (5-HT) on the release of endogenous glutamate (GLU) in rat cerebellum were investigated in slices depolarized with 35 mM K+. The Ca2+-dependent release of GLU was potently inhibited by 5-HT in a concentration-dependent way. Release was also inhibited by the 5-HT1 receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and by the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI). The inhibition by 10 nM 5-HT was partly (35-40%) counteracted by the 5-HT2 receptor antagonist ketanserin but was fully blocked by the mixed 5-HT1/5-HT2 receptor antagonist methiothepin. The effect of 8-OH-DPAT was not affected by ketanserin but was totally antagonized by methiothepin, while the effect of DOI was entirely suppressed by ketanserin. Ketanserin or methiothepin themselves increased (by 23 and 55%, respectively, at 10 nM) the K+-evoked release of GLU. In conclusion the release of endogenous GLU in rat cerebellum can be inhibited by 5-HT through receptors of the 5-HT1 and 5-HT2 type. The enhancement of GLU release by ketanserin or methiothepin could suggest a tonic inhibition. The possible localization of the 5-HT receptors involved in the interaction with the GLU systems is discussed.

Release-Regulating Serotonin 5-HT1D Autoreceptors in Human Cerebral Cortex

Abstract: Release‐regulating 5‐hydroxytryptamine (5‐HT) autoreceptors in the rat brain belong to the 5‐HT1B subtype. On the other hand, the human brain seems to lack 5‐HT1B receptors. In the present work 5‐HT autoreceptors present in human brain were characterized pharmacologically. Synaptosomes prepared from biopsy samples of human neocortex were labeled with [3H]5‐HT and exposed in superfusion to selective 5‐HT receptor agonists and antagonists during K+ depolarization. The rank order of potency of agonists as inhibitors of the [3H]5‐HT overflow was 5‐HT > sumatriptan (5‐HT1D/1B) > 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (5‐HT1A/1D) ≫ 1‐(2,5‐dimethoxy‐4‐iodophenyl)‐2‐aminopropane HCl (5‐HT2/1C). The effect of 5‐HT was insensitive to ketanserin (5‐HT2) but antagonized by methiothepin (5‐HT1/2) or by metergoline (5‐HT1C/1D). The data are compatible with a classification of the human 5‐HT autoreceptor as being of the 5‐HT1D subtype.

Modulation of 5-hydroxytryptamine release by presynaptic inhibitory α2-adrenoceptors in the human cerebral cortex

SummarySlices and synaptosomes from human cerebral cortex (which had to be removed to reach deeply located tumours) and, for comparison, synaptosomes from guinea-pig and rat cerebral cortex were preincubated with [3H]5-hydroxytryptamine and superfused with physiological salt solution containing an inhibitor of 5-hydroxytryptamine uptake. The effects of α-adrenoceptor agaonists and antagonists on the electrically (slices) or potassium-evoked (synaptosomes) tritium overflow were studied.In human cerebral cortical slices, the electrically-evoked [3H] overflow was inhibited by noradrenaline (pIC25 value: 6.35); the non-selective α-adrenoceptor antagonist phentolamine, at a concentration of 0.32 μmol/l, strongly antagonized the inhibitory effect of noradrenaline (apparent pA2 value: 8.19) but did not affect the evoked overflow by itself. In synaptosomes from humans, guinea-pigs and rats, noradrenaline also inhibited the K+-evoked[3H] overflow in a concentration dependent manner; the α2-adrenoceptor clonidine (1 μmol/l), but not the α1-adrenoceptor agonist methoxamine (1 μmol/l), mimicked the effects of noradrenaline; the effect of noradrenaline (0.3 μmol/l) was abolished by the α2-but not by the α1-adrenoceptor antagonist prazosin (1 μmol/l).It is concluded that release-inhibiting adrenoceptors of the α2-subtype exist on 5-hydrpxytryptamine terminals innervating the cerebral cortex in human and guinea-pig brain.

Sensitivity to selective adenosine A1 and A2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices.

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A(1) and A(2A) receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A(1) and A(2A) receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [(3)H]D-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca(2+) ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [(3)H]D-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor DL-TBOA prevented the ischemia-evoked efflux of [(3)H]D-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [(3)H]D-aspartate or glutamate was increased by the A(1) receptor antagonist DPCPX. The A(2A) antagonist SCH 58261 decreased [(3)H]D-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC(50): 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A(1) or the A(2A) receptor antagonist was found on the ischemia-evoked efflux of [(3)H]D-aspartate in Ca(2+)-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A(1) and stimulatory A(2A) receptors that down- or up-regulate the vesicular-like component of glutamate release.