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

Summary Slices 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 -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 -adrenoceptor antagonist phentolamine, at a concentration of 0.32 mol/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 ₂-adrenoceptor clonidine (1 mol/l), but not the ₁-adrenoceptor agonist methoxamine (1 mol/l), mimicked the effects of noradrenaline; the effect of noradrenaline (0.3 mol/l) was abolished by the ₂-but not by the ₁-adrenoceptor antagonist prazosin (1 mol/l).It is concluded that release-inhibiting adrenoceptors of the ₂-subtype exist on 5-hydrpxytryptamine terminals innervating the cerebral cortex in human and guinea-pig brain.Send offprint requests to M. Raiteri at the above address     

Differences in ligand binding profiles between cloned rabbit and human 5-HT1Dα and 5-HT1Dβ receptors: ketanserin and methiothepin distinguish rabbit 5-HT1D receptor subtypes

The study of serotonin receptor function has been complicated by the extreme molecular diversity of serotonin receptor subtypes, the lack of selective agonists and antagonists for many of the subtypes, and divergence in the pharmacological properties of a single receptor subtype across different animal species. An example of this pharmacological diversity between species homologues is provided by the 5-HT_1D receptor subfamily. To further advance the ability to characterize and pharmacologically compare functional responses mediated by native 5-HT_1D receptors, we have cloned the 5-HT_1D and 5-HT_1D receptor subtypes from the rabbit and evaluated their pharmacological profiles using radioligand binding assays. The deduced amino acid sequences of the rabbit 5-HT_1D and 5-HT_1D receptor genes displayed 60% overall identity [75% transmembrane (TM) identity] to each other and > 90% overall identity (95% TM identity) to their corresponding human homologues. Two compounds were identified in binding assays which discriminated between the closely-related 5-HT_1D receptors. Ketanserin exhibited high affinity (pK_i = 7.66) and selectivity ( > 20-fold) for the 5-HT_1D receptor while methiothepin displayed high affinity (pK_i = 7.86) and selectivity (16-fold) for the 5-HT_1D receptor subtype. The rabbit and human recombinant 5-HT_1D receptors showed significant intraspecies (rabbit 5-HT_1D vs. 5HT_1D) and interspecies (i.e. rabbit vs. human 5-HT_1D) similarities in their ligand binding profiles. These data suggest that 5-HT_1D-mediated responses in rabbit preparations may provide information relevant to the pharmacology of the 5-HT_1D receptor subtypes in humans.     

Pharmacological characterizations of recombinant human 5-HT1D1Dα and 5-HT1Dβ receptor subtypes coupled to adenylate cyclase inhibition in clonal cell lines: apparent differences in drug intrinsic efficacies between human 5-HT1D subtypes

Recombinant human 5-HT_1D and 5-HT_1D receptor subtypes were stably expressed in NIH-3T3 fibroblasts (1D cell line) and Y-1 adrenocortical tumor cells (1D cell line), respectively, for pharmacological evaluations of serotonergic compounds to inhibit forskolin-stimulated CAMP accumulation (FSCA). [³H]LSD saturation studies indicated that 5-HT_1D receptor expression levels were slightly higher in the 1D cell line (B _max = 1334 ± 134 fmol/mg protein) than in the (1D) cell line (B _max = 900 ± 900 fmol/mg protein). 5-HT inhibited FSCA with similar potencies (EC₅₀ 2 nM) in both assay systems. The rank order of agonist potencies in both clonal cell lines matched their pharmacological profiles previously determined in binding studies: dihydroergotamine >- 5-carboxamidotryptamine (5-CT) > LSD >- 5-HT > sumatriptan > 1-naphthylpiperazine (1-NP) > yohimbine > 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH DPAT) > 1-(2,5-dimethoxy4-iodophenyl)-2-aminopropane (DOI), with K_i/EC₅₀ ratios greater than unity. Methiothepin acted as a silent antagonist at both human 5-HT_1D and 5-HT_1D receptors with apparent dissociation constants (K_b values) of 12 ± 1 nM and 3 ± 1 nM, respectively. Whereas GR 127,935, metergoline, DOI, and quipazine acted as full agonists in the 1D cell line, these compounds behaved as partial agonists in the 1D cell line.To determine whether high levels of receptor reserve might mask partial agonist activity in the two second messenger assay systems, studies were performed using the irreversible receptor alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). The relationships between receptor occupancy and inhibition of FSCA were determined for 5-HT, sumatriptan, and 1-NP in both clonal cell lines after partial receptor inactivation using Furchgott analysis. Hyperbolic relationships between receptor occupancy and second messenger response were determined for 5-HT in both transfected cell lines. Steep hyperbolic relationships were also found for sumatriptan and 1-NP in the 1D cell line whereas nearly linear relationships were observed for these two compounds in the 1D cell line. Moreover, K_A/EC₅₀ ratios of these compounds were significantly larger in the (1D)(10–32) as compared to the 1D (0.9–2.5) cell line. These data are consistent with the hypothesis that the two heterologous expression systems contain a differential amount of receptor reserve. Despite the presence of an apparently larger receptor reserve in the 1D cell line, GR 127,935, metergoline, DOI, and quipazine behaved as partial agonists.Although the potencies (EC₅₀ values) of compounds matched their respective affinity constants (K_i values) for the closely-related 5-HT_1D subtypes, differences in intrinsic activities were observed for a few compounds between the two 5-HT_1D receptor expression systems. Since receptor reserve is dependent on the properties of both the assay system and drug, the observed variations in intrinsic activity, although influenced by the variable amounts of receptor reserve in the two transfected cell lines, reflect primarily system-independent differences in the intrinsic efficacy of the tested compounds at the two human 5-HT_1D receptors. Higher intrinsic efficacies of compounds at the human 5-HT_1D receptor relative to the human 5-HT_1D subtype may be responsible for the higher intrinsic activities observed in the (1D) cell line, even though receptor reserve is apparently lower in this system.Abbreviations CRC Concentration-response curve - FSCA forskolin-stimulated cAMP accumulation - KA pseudo-dissociation constants - 5-CT 5-carboxamidotryptamine - 5-HT 5-hydroxytryptamine - 5MeOT 5-methoxytryptamine - PAPP 1-[2-(4-aminophenyl) ethyl] -4-(3-trifluoromethylphenyl)-piperazine - 1-NP 1-(1-naphthyl) piperazine - 8-OH-DPAT 8-hydroxy-2-(di-n-propylamino) tetralin - DOI 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane - MK-462 (N,N-dimethyl-2-[5-(1, 2, 4-triazol-l-yl methyl)-1H-indole3-yl]ethylamine - GR 127,935 (2-methyl-4-(5-methyl-[1, 2, 4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide) - GR 46611 5-[4-methoxybenzyl ethylene]-1H-indole3-yl]ethyl amine) - L-694,247 (2-[5-[3-(4-methylsulfonylamino)benzyl-1, 2, 4-oxadiazol-5-yl]-1H-indole3-yl]ethylamine)     

Anxiolytic effects of 5-HT₁A receptors and anxiogenic effects of 5-HT₂C receptors in the amygdala of mice

The aim of the present study is to test a hypothesis that 5-HT(1A) and 5-HT(2C) receptors in the amygdala play an important role in the regulation of anxiety behaviors. We examined alterations in anxiety-like behaviors after manipulation of the expression of 5-HT(1A) and 5-HT(2C) receptors in the amygdala using recombinant adenovirus approaches. Recombinant adenoviruses containing a 5-HT(1A) promoter-controlled 5-HT(1A) antisense sequence or a 5-HT(2C) promoter-controlled 5-HT(2C) sense sequence were injected into the amygdala. Elevated plus-maze (EPM) and open field tests were conducted to determine anxiety-like behavior and locomotor activity. Reductions in the expression of 5-HT(1A) receptors in the amygdala significantly attenuated the time spent in the open arms of EPM and time spent in the center of an open field. Reduction in the percent of time spent in the open arms of EPM is negatively correlated with the density of 5-HT(1A) receptors in the central amygdala. On the other hand, increased expression of 5-HT(2C) receptors reduced the time spent in the open arms of EPM and time spent in the center of an open field. The reductions in the time spent and distance traveled in the open arms of EPM were correlated to the density of 5-HT(2C) receptors in the basolateral nucleus of amygdala. These data suggest that amygdaloid 5-HT(1A) receptors produce anxiolytic and 5-HT(2C) receptors produce anxiogenic effects. Together, the present results demonstrate the important role of the amygdaloid 5-HT(1A) and 5-HT(2C) receptors in the regulation of anxiety-like behaviors. This article is part of a Special Issue entitled 'Anxiety and Depression'.     

Involvement of 5-HT₇ receptors in the pathogenesis of temporal lobe epilepsy

The 5-hydroxytryptamine 7 (5-HT(7)) receptor is the most recently classified member of the serotonin receptor family. The localization of 5-HT(7) receptors and the biological activity of its ligands have suggested that 5-HT(7) receptors might be involved in the pathogenesis of epilepsy. In the present study, we investigated the correlation between temporal lobe epilepsy and 5-HT(7) receptors using pilocarpine-induced rat models of temporal lobe epilepsy and surgical samples of temporal neocortex from intractable epilepsy patients. An analysis of electroencephalogram (EEG) and behavioral changes before and after the treatment of SB269970 hydrochloride (a selective 5-HT(7) receptor antagonist, 10 mg/kg, i.p.) and AS19 (a selective 5-HT(7) receptor agonist, 10 mg/kg, s.c.) demonstrated that in epileptic rats the activation of 5-HT(7) receptors could increase the number of seizures, which could be reduced by a 5-HT(7) receptor antagonist. Moreover, the expression of 5-HT(7) receptors was higher in the epilepsy group compared with the nonepileptic group in both rat and human brain tissues. The present results suggested that 5-HT(7) receptors participate in the pathogenesis of temporal lobe epilepsy, and a 5-HT(7) receptor antagonist may be used as a therapeutic alternative for temporal lobe epilepsy.     

Facilitation of serotonin (5-HT) release in the rat brain cortex by cAMP and probable inhibition of adenylate cyclase in 5-HT nerve terminals by presynaptic α2-adrenoceptors

Summary Stimulation-evoked tritium overflow was examined in superfused rat brain cortex slices (stimulus: electrical impulses; 3 Hz) and synaptosomes (stimulus: potassium 12 mmol/l) preincubated with ³H-5-HT. 1. In slices and synaptosomes, the evoked ³H overflow was facilitated by forskolin and 8-Br-cAMP, but was not affected by AH 21-132 (an inhibitor of cAMP phosphodiesterase; cis-6-(p-acetamidophenyl)-1, 2, 3, 4, 4 a,10b-hexahydro-8, 9-dimethoxy-2-methylbenzo [c] [1,6]-naphthyridine). In the presence of AH 21-132, the facilitatory effect of forskolin on evoked overflow was increased. 2. In slices, AH 21-132 or combined administration of forskolin plus AH 21-132 did not change the percentage of basal or evoked ³H overflow represented by unmetabolized ³H-serotonin (about 30% and 60%, respectively). 3. In slices, cocaine or 6-nitroquipazine, an inhibitor of serotonin uptake, did not influence the increase in evoked overflow produced by forskolin plus AH 21-132. Forskolin plus AH 21-132 did not alter the inhibitory effect of serotonin (examined in the presence of 6-nitroquipazine) and the facilitatory effect of metitepin (a serotonin receptor antagonist) on evoked ³H overflow, but considerably decreased the inhibitory effect of clonidine or B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[5,4-d]-azepine). The present results suggest that the serotoninergic nerve terminals in the rat brain cortex are endowed with an adenylate cyclase, which is negatively coupled to the presynaptic ₂-adrenoceptors, but is not linked to the presynaptic autoreceptors.This study was supported by a grant of the Deutsche Forschungsgemeinschaft. Part of the present results was reported at the 9th International Congress of Pharmacology, London 1984 (Schlicker et al. 1984) Send offprint requests to M. Göthert     

5-HT1-like receptor mediated changes in porcine carotid haemodynamics: are 5-HT1D receptors involved?

Summary 5-Hydroxytryptamine (5-HT) reduces porcine arteriovenous shunting in the carotid vascular bed by stimulation of both 5-HT₁-like and 5-HT₂ receptors and increases capillary flow to some tissues, like the skin and ears, by different 5-HT₁-like receptors. In view of the heterogeneous nature of the 5-HT₁-like receptors and the relative selectivity for the 5-HT_1D binding sites of sumatriptan, which also reduces porcine arteriovenous shunting and slightly increases capillary blood flow towards skin and ears by 5-HT₁-like receptors, we have attempted to determine whether one or both of these carotid 5-HT₁-like receptors belong to the 5-HTID subtype.Pentobarbitone anaesthetized pigs, subjected to bilateral cervical vagosympathectomy, received either 5-HT (2 g · kg^–1 · min^–1) in the carotid artery or cumulative i.v. doses of sumatriptan (10, 30, 100 and 300 g · kg^–1). Their effect on the total carotid blood flow and its distribution into capillary and arteriovenous anastomotic parts was determined with radioactive microspheres. The effect of metergoline (1 mg·kg^–1), a substance with a very high affinity for the 5-HT_1D receptor as well as for the 5-HT_1A, 5-HT_1B, 5-HT_1C and 5-HT₂ receptors, was studied on the responses to 5-HT and sumatriptan.Both 5-HT and sumatriptan reduced carotid arteriovenous anastomotic blood flow. 5-HT and, to a lesser extent, sumatriptan also increased capillary blood flow towards some tissues. Metergoline by itself did not affect the distribution of porcine carotid blood flow. It attenuated the constrictor response, but increased the vasodilator response to 5-HT, in a manner similar to the 5-HT₂ receptor antagonists cyproheptadine, ketanserin and WAL 1307 in our former experiments. These effects seem, therefore, to be related to the blockade of 5-HT₂ receptors by metergoline. On the other hand, metergoline had no significant effect against the responses to sumatriptan.It is concluded that neither the constrictor nor the dilator carotid 5-HT₁-like receptors seem to be related to the known 5-HT₁ binding subtypes, including the 5-HT_1D subtype. Send offprint requests to M. O. Den Boer at the above address     

β-cypermethrin-induced acute neurotoxicity in the cerebral cortex of mice

A Type II pyrethroid pesticide β-cypermethrin is widely used in agriculture and domestic applications for pest control. However, the effect of β-cypermethrin on the glutamate neurotransmitter has not been well-documented. In the current study, mice were treated with 20, 40, or 80?mg/kg β-cypermethrin by a single oral gavage, with corn oil as a vehicle control. Four hours after treatment, we investigated glutamate levels and glutamate-metabolizing enzyme (phosphate-activated glutaminase, PAG; glutamine synthetase, GS) activities in the cerebral cortex of mice, using a HPLC system with ultraviolet detectors and a colorimetric assay. Glutamate uptake levels in the synaptosomes of cerebral cortex and mRNA expression levels of PAG, GS, and glutamate transporter-1 (GLT-1) in the cerebral cortex were detected by a radioactive labeling method and qRT-PCR, respectively. Toxic symptoms were observed in mice treated with 40 or 80?mg/kg β-cypermethrin. Compared with the control, significant decreases in glutamate level and GS activity, and an obvious increase in synaptosomal glutamate uptake, were found in the cerebral cortex of mice treated with 80?mg/kg β-cypermethrin. No significant changes were found among groups in PAG activity or PAG, GS, and GLT-1 mRNA expression levels. These results suggest that β-cypermethrin treatment may reduce the glutamate level in the mouse cerebral cortex, which is associated with decreased GS activity and increased synaptosomal glutamate uptake. Our findings provide a partial explanation for the neurotoxic effects of synthetic β-cypermethrin insecticides.     

Comparison of the densities of 5-HT4 receptors, β1- and β2-adrenoceptors in human atrium: functional implications

We measured in human atrium the density of 5-HT₄ receptors, labelled with [¹²⁵I]-SB 207710 (1-butyl-4-piperidinyl) methyl 8-amino-7-iodo-1, (4-benzodioxan-5-carboxylate), and compared it with the density of ₁- and ₂-adrenoceptors, labelled with (–)-[¹²⁵I]-cyanopindolol. [¹²⁵I]-SB 207710 (5–1200 pmol/l) labelled a small population of saturable binding sites (B _max 4 fmol/mg protein) with a pK_D of 9.7 and with 5-HT₄ receptor characteristics, as assessed with competing ligands. The density of atrial binding sites with 5-HT₄ receptor characteristics was 10 and 5 times lower, respectively, than the density of ₁- and ₂-adrenoceptors. We suggest that the small 5-HT₄ receptor population may in part explain why the positive inotropic effects of 5-HT are smaller than those of catecholamines mediated through ₁- and ₂-adrenoceptors.     

Mutual interaction between presynaptic α2-adrenoceptors and 5-HT1B receptors on the sympathetic nerve terminals of the rat inferior vena cava

Summary Segments of the rat vena cava preincubated with [³H]noradrenaline and superfused with [³H]noradrenaline-free solution containing desipramine and corticosterone were stimulated electrically (standard parameters: 150 mA, 0.3 ms, 0.66 Hz; duration 6 min). In some experiments the stimulation parameters were modified in order to obtain similar absolute release values despite the presence of an -adrenoceptor agonist or antagonist or of 5-hydroxytryptamine (5-HT).In a first set of experiments, the vascular segments were first exposed to an -adrenoceptor ligand, which was kept present throughout the remainder of superfusion, and then to 5-HT. The release-inhibiting effect of 5-HT was attenuated by the ₂-adrenoceptor agonists clonidine and B-HT 920 whereas it was enhanced by the -adrenoceptor antagonists phentolamine and idazoxan. The ₁-adrenoceptor antagonist prazosin did not change the 5-HT-induced inhibition of noradrenaline (NA) release. In a second set of experiments, 5-HT was administered first and kept present in the superfusion fluid for the remainder of the experiment. In the presence of 5-HT, the overflow-inhibiting effects of B-HT 920 and clonidine and the overflow-enhancing effect of idazoxan were reduced.The results demonstrate that activation of one kind of receptor decreased the inhibition of noradrenaline release produced by activation of the other. These effects were not the consequence of the change of release per se induced by the interacting drugs, since they also occurred when the release was adjusted to similar levels by modification of the stimulation parameters. Therefore, the effects are probably due to mutual interaction between presynaptic ₂-adrenoceptors and 5-HT_1B receptors. The level (receptors themselves, G proteins and/or ion channels) at which this interaction occurs is unknown. Send offprint requests to M. Göthert at the above address     

How selective is GR 43175? Interactions with functional 5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D receptors

Summary GR 43175 (3-[2-dimethylamino]ethyl-N-methyl-1H-indole-5 methane sulphonamide) is a novel 5-HT₁-like receptor-selective agonist which was reported to be active in the treatment of migraine attacks. The effects of the compound were investigated in radioligand binding studies and in functional models for 5-HT_1A, 5-HT_1B, and 5-HT_1D receptors (inhibition of forskolin-stimulated adenylate cyclase activity in calf hippocampus, rat and calf substantia nigra, respectively) and 5-HT_1C receptors (stimulation of inositol phosphate production in pig choroid plexus).GR 43175 displayed the following order of affinity for 5-HT recognition sites (pK_D values, -log mol/l, in parentheses): 5-HT_1D (7.54) > 5-HT_1B (6.35) > 5-HT_1A (6.13) 5-HT_1C (4.13) > 5-HT₂ (3.67). The same order of potency was observed at functional 5-HT₁ receptors, at which GR 43175 acted as a full agonist, with the exception of the 5-HT_1C receptor, where the compound was a weak antagonist (pEC₅₀ or pK_B values, -log mol/l, in parentheses): 5-HT_1D (6.28) > 5-HT_1B (6.03) > 5-HT_1A (5.57) > 5-HT_1C (4.25).The present data show that GR 43175 interacts preferentially as an agonist with 5-HT_1B and 5-HT_1D receptors. Since 5-HT_1B receptors have not yet been identified in human brain, it seems possible that it is the 5-HT_1D receptor which is relevant to the reported antimigraine effects of this compound.Send offprint requests to D. Hoyer at the above address     

“5-HT1R” or 5-HT1D sites? Evidence for 5-HT 1D binding sites in rabbit brain

Summary Radioligand binding studies were performed in membranes of rabbit whole brain and striatum using the novel iodinated radioligand for 5-hydroxytryptamine 5-HT_1B and 5-HT_1D sites, Serotonin-5-O-Carboxymethyl-Glycyl[¹²⁵I]Tyrosinamide ([¹²⁵I]GTI).[¹²⁵I]GTI labelled a finite number of high affinity sites in rabbit brain membranes, B_max = 191 +- 47 fmol/mg protein, pK_D (-log mol/1) = 8.50 +- 0.13, n = 5. The pharmacological profile of [¹⁵²I]GTI binding was fully comparable to that reported previously in human and other brain preparations known to possess 5-HT_1D sites (using either [³H]5-HT or [¹²⁵I]GTI) and displayed a characteristic rank order of affinity: 5-carboxamidotryptamine > 5-HT = dihydroergotamine _> ergotamine _ sumatriptan >_ CGS 12066 >- metergoline > yohimbine >_ methysergide > ICYP > 8-OH-DPAT >_ CP 93129 > (-)pindolol > ketanserin > isamoltane > mesulergine > corynanthine > buspirone > MDL 72222.Autoradiographic studies were performed on rabbit brain slices using [³H]5-HT in the presence of 100 nmol/1 8-OH-DPAT and mesulergine (in order to mask 5-HT_1A and 5-HT_1c binding sites) and [¹²⁵I]CYP (iodocyanopindolol) in the presence of µmol/I isoprenaline and 100 nmol/l 8-OH-DPAT (in order to mask adrenoceptor and 5-HT_1A binding sites). There was no detectable specific binding of [¹²⁵I]CYP through the brain, thus excluding the presence of 5-HT_1B sites in rabbit brain. By contrast, [³H]5-HT labelled a high density of sites in globus pallidus, substantia nigra and superior colliculus. Other regions displaying labelling include the striatum, the dentate gyrus of the hippocampus and the periaqueductal grey matter. This pattern of distribution is compatible with that reported for 5-HT_1D sites in other species.The present data strongly suggest that rabbit brain has recognition sites with the pharmacological profile and distribution characteristic of the 5-HT_1D recognition site. These findings are in agreement with results obtained by Limberger et al. (1991), suggesting the terminal 5-HT autoreceptor of the rabbit brain to belong to the 5-HT_1D subtype. Except for limited species variations (see Bruinvels et al. 1992), the pharmacology of these sites does not justify an addition to the existing nomenclature (5-HT_1R), in contrast to what has been proposed by Xiong and Nelson (1989). The rabbit represents another laboratory species in which 5-HT_1D receptor mediated effects could be studied. Correspondence to D. Hoyer at the above address     

Subclassification of presynaptic α2-adrenoceptors: α2D-autoreceptors in mouse brain

The study was devised to classify, by means of antagonist affinities, the presynaptic ₂-autoreceptors in mouse cerebral cortex in terms of _2A, _2B, _2C and _2D. A set of antagonists was chosen that was able to discriminate between the four subtypes. Slices of the cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically.The stimulation periods used (4 pulses, 100 Hz) did not lead to ₂-autoinhibition as shown by the lack of an increase by rauwolscine of the evoked overflow of tritium. The ₂-selective agonists 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14,304) and -methylnoradrenaline reduced the evoked overflow. All 10 antagonists shifted the concentration-inhibition curve of UK 14,304 to the right. Rauwolscine also shifted the concentration-inhibition curve of -methylnoradrenaline to the right. pK_d values of the antagonists were calculated from the shifts. The pK_d values of rauwolscine against UK 14,304 and -methylnoradrenaline were very similar (8.0 and 7.9, respectively).Comparison with antagonist affinities for prototypic native ₂ binding sites, ₂ binding sites in cells transfected with ₂ subtype genes, and previously classified presynaptic ₂-adrenoceptors — all taken from the literature — indicates that the ₂-autoreceptors in mouse brain cortex are _2D. This is the first subtype determination of ₂-autoreceptors in the mouse. It supports the hypothesis that at least the majority of ₂-autoreceptors belong to the _2A/D branch of the ₂-adrenoceptor tree.     

Signaling pathways leading to phosphorylation of Akt and GSK-3β by activation of cloned human and rat cerebral D₂and D₃ receptors

Although dopamine (DA) regulates the serine/threonine kinase Akt and its downstream substrate glycogen synthase kinase-3β (GSK-3β), the direct influence of dopaminergic receptors remains poorly characterized. Short-term incubation of Chinese hamster ovary (CHO)-expressed human (h)D(?L) and hD?) receptors with DA (maximal effect, 5-10 min) phosphorylated Akt (Thr308 and Ser473) and GSK-3β (Ser9), actions blocked by the selective D? and D? antagonists, 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-l-yl]methyl-1H-indole (L741,626) and (3aR,9bS)-N[4-(8-cyano-1,3a,4,9b-tetrahydro-3H-benzopyrano[3,4-c]pyrrole-2-yl)-butyl] (4-phenyl)benzamide (S33084), respectively. Similar findings were acquired with the specific D?/D? receptor agonist quinelorane, which also enhanced (10 min after administration) levels of p-Akt and p-GSK-3β in rat nucleus accumbens, an action blocked by the D?/D? receptor antagonist raclopride. Akt and GSK-3β phosphorylation mediated via CHO-expressed hD(?L) and hD? receptors was prevented by pertussis toxin and by inhibitors of insulin-like growth factor-1 receptors as well as phosphatidylinositol 3-kinase and Src. Likewise, chelation of intracellular Ca2+ and interference with an "atypical" phorbol ester-insensitive protein kinase C (PKC) abolished recruitment of Akt and GSK-3β. Inactivation of PKCμ blocked Akt and GSK-3β phosphorylation at hD(?L) receptors. However, blockade of conventional PKC isoforms attenuated the actions of DA at hD? receptors only. Furthermore, phospholipase C (PLC), calmodulin, and Akt inhibitors abolished DA-induced GSK-3β phosphorylation by hD? receptors, whereas phosphorylation by hD(?L) receptors partially involved calmodulin, Akt, and extracellular signal-regulated kinase (ERK) 1/2. In conclusion, at both hD(?L) and hD? receptors, DA elicited a G(i/o)- and Ca2+/calmodulin-dependent phosphorylation of Akt and GSK-3β via transactivation of insulin-like growth factor 1 receptor. However, significant differences were seen regarding the involvement of PLC, calmodulin, and ERK1/2.     

The role of 5-HT₁A receptors in fish oil-mediated increased BDNF expression in the rat hippocampus and cortex: a possible antidepressant mechanism

Epidemiological and dietary studies show that nutritional deficit of omega-3 polyunsaturated fatty acids (ω-3 PUFA) is directly related to the prevalence and severity of depression. Supplementation with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) during critical periods of development (pregnancy and lactation) is essential for cortical maturation, synaptogenesis and myelination, and may also mitigate the risk for cognitive deficits and psychopathologies in young adults. The present study was performed to evaluate the involvement of serotonin (5-HT) receptors, particularly of 5-HT(1A), and hippocampal brain-derived neurotrophic factor (BDNF) expression in the antidepressant effect of ω-3 PUFA supplementation. In Experiment 1, the antidepressant effects of fish oil were assessed by the modified forced swim test in adult rats. The data indicated a robust antidepressant effect produced by this supplementation and that treatment of the rats with WAY 100135 reversed this effect. In Experiment 2, cortical and hippocampal contents of BDNF, 5-HT, dopamine (DA) and its metabolites, 5-hydroxyindoleacetic acid (5-HIAA), and 3,4-dihydroxyphenylacetic acid (DOPAC), were determined in animals subjected to the same protocol. Increased BDNF expression in the cortex and hippocampus of both age groups was detected. In 90 day-old rats, 5-HT content in the hippocampus was increased, whereas 5-HIAA formation was diminished in the fish oil group. We suggest the occurrence of a reciprocal involvement of 5-HT(1A) receptors activation and the hippocampal BDNF-increased expression mediated by fish oil supplementation. These data corroborate and expand the notion that supplementation with ω-3 PUFA produces antidepressant effects mediated by an increase in serotonergic neurotransmission, particularly in the hippocampus. This article is part of a Special Issue entitled 'Anxiety and Depression'.     

Radiosynthesis and preclinical evaluation of [11C]Cimbi-701 – Towards the imaging of cerebral 5-HT7 receptors

The serotonin 7 (5-HT₇) receptor is suggested to be involved in a broad variety of CNS disorders, but very few in vivo tools exist to study this important target. Molecular imaging with positron emission tomography (PET) would enable an in vivo characterization of the 5-HT₇ receptor. However, no clinical PET radiotracer exists for this receptor, and thus we aimed to develop such a tracer. In this study, we present the preclinical evaluation of [¹¹C]Cimbi-701. Cimbi-701 was synthesized in a one-step procedure starting from SB-269970. Its selectivity profile was determined using an academic screening platform (NIMH Psychoactive Drug Screening Program). Successful radiolabeling of [¹¹C]Cimbi-701 and subsequent in vivo evaluation was conducted in rats, pigs and baboon. In vivo specificity was investigated by 5-HT₇ and σ receptor blocking studies. P-gp efflux transporter dependency was investigated using elacridar. [¹¹C]Cimbi-701 could successfully be synthesized. Selectivity profiling revealed high affinity for the 5-HT₇ (K_i = 18 nM), σ-1 (K_i = 9.2 nM) and σ-2 (K_i = 1.6 nM) receptors. In rats, [¹¹C]Cimbi-701 acted as a strong P-gp substrate. After P-gp inhibition, rat brain uptake could specifically be blocked by 5-HT₇ and σ receptor ligands. In pig, high brain uptake and specific 5-HT₇ and σ-receptor binding was found for [¹¹C]Cimbi-701 without P-gp inhibition. Finally, low brain uptake was found in baboons. Both the specific σ-receptor binding and the low brain uptake of [¹¹C]Cimbi-701 displayed in baboon discouraged further translation to humans. Instead, we suggest exploration of this structural class as results indicate that selective 5-HT₇ receptor imaging might be possible when more selective non-P-gp substrates are identified.     

The 5-HT1A receptor agonist 8-OH-DPAT reduces rats’ accuracy of attentional performance and enhances impulsive responding in a five-choice serial reaction time task: role of presynaptic 5-HT1A receptors

Rationale: Whilst several studies have investigated the role of serotonergic receptor subtypes in learning and memory, relatively few studies have examined their role in attentional processes. Objective: The present study investigated the role of pre- and postsynaptic 5-HT_1A receptors on rats’ attentional performance in the five-choice serial reaction time task (5-CSRT). Methods: Hungry rats were trained in the 5-CSRT task to detect brief (0.5 s) flashes of light presented randomly in one of five locations with a fixed intertrial interval of 5 s paced by the rat. We studied the effects of 8-OH-DPAT, a 5-HT_1A receptor agonist, at various subcutaneous (SC) doses (10–100 μg/kg) on measures of rats’ discriminative accuracy (the index of attentional functioning) and various behavioural indices of response control and motivation. Manipulations of basic task parameters, intracerebroventricular (ICV) injections of 5,7-dihydroxytryptamine (5,7-DHT) to deplete forebrain 5-HT and treatments with a selective 5-HT_1A receptor antagonist WAY 100635 were made in order to determine the behavioural and neural specificity of the effects of 8-OH-DPAT. Results: A dose of 100 μg/kg, but not lower doses, significantly reduced choice accuracy and increased errors of omission, latencies to respond correctly and to collect food reward and premature responses. All these effects were completely blocked by WAY 100635, injected SC 5 min before 8-OH-DPAT at doses from 10–100 μg/kg. WAY 100635 by itself had no effect in the task. Dimming the visual stimuli to one-third of the usual brightness did not modify the effect of 8-OH-DPAT on choice accuracy. Prolonging the stimuli from 0.5 to 1.0 s reversed 8-OH-DPAT’s effect on choice accuracy but did not modify the other effects on rats’ performance. An ICV injection of 150 μg 5,7-DHT, which depleted forebrain serotonin by 90%, reversed 8-OH-DPAT’s effect on choice accuracy but did not modify the effects on errors of omission and latency to make correct responses. Similar effects were found by infusing 1.0 μg/0.5 μl WAY 100635 in the dorsal raphe 5 min before 8-OH-DPAT. 8-OH-DPAT increased the latency to collect the reinforcement; this effect was attenuated by ICV 5,7-DHT and completely antagonized by WAY 100635 in the dorsal raphe. Rats treated with 5,7-DHT or 8-OH-DPAT showed more premature responses and these effects were markedly reduced by the combined treatment. Conclusions: The results suggest that stimulation of presynaptic 5-HT_1A receptors is involved in the ability of 8-OH-DPAT to cause attentional dysfunction and enhance impulsivity while slowing of responding and increase in errors of omission mainly depend on stimulation of postsynaptic 5-HT_1A receptors. Received: 7 August 1999 / Final version: 14 December 1999     

Structural analysis by the comparative molecular field analysis method of the affinity of β-adrenoreceptor blocking agents for 5-HT1A and 5-HT1B receptors

The affinities of 17β-adrenoreceptor antagonists for 5-HT_1A and 5-HT_1B receptors were evaluated in binding assays. A large range of K_i values (2–10,000 nM) was observed and ortho or meta substitution of the aromatic ring carrying the amino chain was implicated in the high affinity K_i values, whereas para substitution elicited a dramatic drop in activity. These variations were analyzed with two molecular design tools: the active analogue approach (AAA) and the new 3D-QSAR (quantitative structure activity relationship) method, comparative molecular field analysis (CoMFA). The AAA method emphasized, by superimposition of selected conformations of the molecules, the favorable and unfavorable volumes implicated in the receptor recognition. CoMFA generated a linear expression between the biological data and the different values of electrostatic and steric fields surrounding the molecules. It predicted the values of selected molecules but also those of new molecules not included in the study. The excellent accuracy of the prediction revealed the potential of the method for the design of new compounds. CoMFA demonstrated the important contribution of steric parameters, evaluated at 92%, compared to the electrostatic field (evaluated at 8%) to explain the affinity for 5-HT_1A and 5-HT_1B receptors. This study emphasizes also the importance of the occupancy of a hydrophobic pocket in the receptor site located near the area interacting with the aromatic moiety, and subsequently its use for the design of new, potent, specific antagonists of 5-HT_1A and 5-HT_1B receptors.     

Mutual independence of 5-HT2 and α1 noradrenergic receptors in mediating deficits in sensorimotor gating

Rationale  

Prepulse inhibition (PPI), a preattentional information-filtering mechanism, is disrupted by serotonin (5-HT) or norepinephrine (NE) agonists to model deficits seen in schizophrenia, but whether this effect occurs through interactions between these systems is not known.