Repeated administration of the 5-HT<Subscript>1B</Subscript> receptor antagonist SB-224289 blocks the desensitisation of 5-HT<Subscript>1B</Subscript> autoreceptors induced by fluoxetine in rat frontal cortex

Desensitisation of 5-HT_1A and 5-HT_1B autoreceptors is thought to be the mechanism underlying the therapeutic effects of fluoxetine and other selective serotonin re-uptake inhibitors (SSRIs) when these are administered chronically, while blockade of these autoreceptors occurring on administration of an SSRI together with an autoreceptor antagonist is responsible for the acute increase in 5-HT levels in vivo observed under these circumstances. The effects of repeated administration of SSRIs together with 5-HT_1B receptor antagonists on 5-HT levels and autoreceptor activity have not been studied previously with an in vivo method. In this work we found, using in vivo microdialysis that the effect of fluoxetine (5 mg/kg i.p. daily for 7 days) to desensitise 5-HT_1B autoreceptors in frontal cortex, as measured by the action of CP 93129 (10 M) to reduce 5-HT levels, was prevented by concomitant administration of the 5-HT_1B receptor antagonist SB 224289 (2.5 mg/kg s.c.). 5-HT_1B receptor activity in hypothalamus and 5-HT_1A autoreceptor activity, as determined by the effects of s.c. 8-OH-DPAT to reduce 5-HT levels, were not altered either by fluoxetine alone at this dose or by fluoxetine in the presence of SB 224289. We conclude that the effects obtained when 5-HT_1B autoreceptor antagonists are administered acutely together with SSRIs may not be maintained after repeated administration.     

Role of extracellular serotonin levels in the effect of 5-HT<Subscript>1B</Subscript> receptor blockade

The release of serotonin (5-HT) at serotonergic nerve terminals is regulated by 5-HT_1B autoreceptors. Several studies have reported that the effects of selective 5-HT reuptake inhibitors (SSRIs) on extracellular 5-HT are augmented by 5-HT_1B receptor antagonists, whereas administration of these antagonists alone do not enhance 5-HT levels. It has been suggested that 5-HT_1B receptors have low basal endogenous activity and therefore elevated endogenous 5-HT levels are needed to elicit an effect of 5-HT_1B receptor antagonists. To test this hypothesis, different strategies were used to enhance 5-HT levels in the rat frontal cortex to assess the effects of locally applied NAS-181, a new selective 5-HT_1B receptor antagonist. Blockade of 5-HT_1B receptors with NAS-181 dose dependently augmented 5-HT levels when 5-HT levels were enhanced by a SSRI. No additional effect of NAS-181 on 5-HT output was found when 5-HT levels were enhanced by KCl depolarization-induced release or by preventing degradation of 5-HT with the monoamine oxidase inhibitor pargyline. In the presence of fluvoxamine, the increased 5-HT release evoked by KCl depolarization was augmented by NAS-181, supporting the idea that blockade of 5-HT transporters is necessary to measure an effect of 5-HT_1B receptor blockade. In conclusion, the results provide circumstantial evidence that the effect of a 5-HT_1B receptor antagonist depends on extracellular 5-HT levels, but strongly suggest that additional 5-HT reuptake inhibition is required to detect any effect of 5-HT_1B receptor antagonist on 5-HT levels by in vivo microdialysis. Electronic Publication     

Differential autoreceptor control of extracellular 5-HT in guinea pig and rat: species and regional differences

Rationale Central 5-hydroxytryptamine (5-HT) release is regulated by inhibitory 5-HT autoreceptors, including 5-HT_1A and 5-HT_1B receptors.Objectives The purpose of this study was to use combinations of selective autoreceptor antagonists to elucidate the role of these receptors in controlling extracellular 5-HT in terminal areas.Methods Microdialysis was carried out in awake rats and guinea pigs to measure extracellular 5-HT in the frontal cortex and dentate gyrus. Using the selective 5-HT_1A receptor antagonist, WAY-100635, and the selective 5-HT_1B receptor antagonist, SB-224289, we have compared the roles of 5-HT_1A and 5-HT_1B autoreceptors in controlling extracellular 5-HT.Results SB-224289 (4 mg/kg i.p.) alone produced a significant 50% increase in extracellular 5-HT in the dentate gyrus of guinea pigs, but not in the frontal cortex of the same animals. Co-administration of WAY-100635 (0.3 mg/kg s.c.), did not change the SB-224289-induced increase in dentate gyrus 5-HT but did produce a significant augmentation (60% increase) of guinea pig frontal cortex 5-HT. In contrast, neither autoreceptor antagonist, alone or in combination, affected extracellular 5-HT in the frontal cortex or dentate gyrus of rats.Conclusions These data indicate that there is a species difference in the autoreceptor control of 5-HT release. Furthermore, in the guinea pig there is a divergence between dorsal and median raphe innervated brain regions. On the basis that antagonism of 5-HT_1A and 5-HT_1B receptors produced an immediate increase in extracellular 5-HT in multiple brain regions in the guinea pig, it is suggested that this might be a novel mechanism for achieving antidepressant efficacy.     

Characterization of the potent 5-HT(1A/B) receptor antagonist and serotonin reuptake inhibitor SB-649915: preclinical evidence for hastened onset of antidepressant/anxiolytic efficacy

An increase in brain serotonin (5-HT) levels is thought to be a key mechanism of action responsible for generating antidepressant efficacy. It has been proven that selective serotonin reuptake inhibitors are effective antidepressants, but the delay to therapeutic onset of these agents is thought to be due to the time required for 5-HT1A, and possibly 5-HT1B, autoreceptors to desensitize. Therefore, an agent incorporating 5-HT reuptake inhibition coupled with 5-HT1A and/or 5-HT1B autoreceptor antagonism may provide a fast-acting clinical agent. The current studies review the profile of SB-649915 (6-[(1-{2-[(2-methylquinolin-5-yl)oxy]ethyl}piperidin-4-yl)methyl]-2H-1,4-benzoxazin-3(4H)-one), a novel compound with high affinity for human (h) 5-HT1A and 5-HT1B receptors (pKi values of 8.6 and 8.0, respectively) as well as the (h) 5-HT transporter (SERT) (pKi value of 9.3). SB-649915 behaved as an antagonist at both 5-HT1A and 5-HT1B receptors in vitro and in vivo, reversing 5-HT, (+)8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) and SKF99101-induced functional/behavioral responses. Furthermore, it inhibited [3H]5-HT reuptake in rat cortical synaptosomes, in vitro and ex vivo. In electrophysiological studies SB-649915 had no effect on rat dorsal raphe neuronal cell firing per se, but reversed 8-OH-DPAT-induced inhibition of firing both in vitro and in vivo. In addition, in a microdialysis study, it produced an acute increase in extracellular 5-HT in forebrain structures of the rat. Finally, SB-649915 demonstrated acute anxiolytic activity in both rodent and non-human primate and reduced the latency to onset of anxiolytic behavior, compared to paroxetine, in the rat social interaction paradigm. In summary, SB-649915 is a novel, potent 5-HT1A/1B autoreceptor antagonist, and 5-HT reuptake inhibitor. This particular pharmacological profile provides a novel mechanism that could offer fast-acting antidepressant activity.     

Role of 5-HT<Subscript>2A</Subscript> and 5-HT<Subscript>2C/B</Subscript> receptors in the acute effects of 3,4-methylenedioxymethamphetamine (MDMA) on striatal single-unit activity and locomotion in freely moving rats

Rationale Like amphetamine, a locomotor-activating dose of 3,4-methylenedioxymethamphetamine (MDMA) predominantly excites striatal single-unit activity in freely moving rats. Although both D₁- and D₂-like dopamine (DA) receptors play important roles in this effect, MDMA, unlike amphetamine, strongly increases both DA and serotonin (5-HT) transmission. Objectives This study was conducted to investigate the 5-HT receptor mechanisms underlying the striatal effects of MDMA. Methods We recorded the activity of >200 single units in the striatum of awake, unrestrained rats in response to acute MDMA administration (5 mg/kg) combined with the selective blockade of either 5-HT_2A or 5-HT_2C/B receptors. Results Prior administration of SR-46349B (a 5-HT_2A antagonist 0.5 mg/kg) blocked nearly all MDMA-induced striatal excitations, which paralleled its significant attenuation of MDMA-induced locomotor activation. Conversely, prior administration of SB-206553 (a 5-HT_2C/B antagonist 2.0 mg/kg) had no effect on the amount of MDMA-induced locomotor activation or the distribution of single-unit responses to MDMA. However, a coefficient-of-variation analysis indicated significantly less variability in the magnitude of both MDMA-induced neuronal excitations and inhibitions in rats that were pretreated with SB-206553 compared to vehicle. Analysis of concurrent single-unit activity and behavior confirmed that MDMA-induced striatal activation was not merely due to behavioral feedback, indicating a primary action of MDMA. Conclusion These results support and extend our previous findings by showing that 5-HT_2A and 5-HT_2C/B receptors differentially regulate the expression of MDMA-induced behavioral and striatal neuronal responses, either directly or through the modulation of DA transmission.     

SB-649915, a novel, potent 5-HT1A and 5-HT1B autoreceptor antagonist and 5-HT re-uptake inhibitor in native tissue

An increase in brain 5-HT levels is thought to be the key mechanism of action which results in an antidepressant response. It has been proven that selective serotonin re-uptake inhibitors are effective antidepressants but the delay to therapeutic onset of these agents is thought to be due to the time required for 5-HT1A, and possibly 5-HT1B, autoreceptor desensitisation. Therefore an agent incorporating 5-HT re-uptake inhibition coupled with 5-HT1A and/or 5-HT1B autoreceptor antagonism may provide a fast acting clinical agent. The current studies describe the in vitro profile of SB-649915 (6-[(1-{2-[(2-methylquinolin-5-yl)oxy]ethyl}piperidin-4-yl)methyl]-2H-1,4-benzoxazin-3(4H)-one), a novel compound which has high affinity for human recombinant 5-HT1A, 5-HT1B and 5-HT1D receptors (pKi values of 8.6, 8.0, 8.8, respectively) and the human recombinant 5-HT transporter (pKi value of 9.3). SB-649915 also displays high affinity for rat, guinea pig, mouse and marmoset native tissue 5-HT1A, 5-HT1B and 5-HT1D receptors and rat native tissue 5-HT transporters (pKi values>or=7.5). In functional [35S]GTPgammaS binding studies, SB-649915 (up to 1 microM) does not display intrinsic activity in HEK293 cells expressing human recombinant 5-HT1A receptors but acts as a partial agonist at human recombinant 5-HT1B and 5-HT1D receptors with intrinsic activity values of 0.3 and 0.7, respectively, as compared to the full agonist 5-HT. From Schild analysis, SB-649915 caused a concentration-dependent, rightward shift of 5-HT-induced stimulation of basal [35S]GTPgammaS binding in cells expressing human recombinant 5-HT1A or 5-HT1B receptors to yield pA2 values of 9.0 and 7.9, respectively. In electrophysiological studies in rat dorsal raphe nucleus, SB-649915 did not affect the cell firing rate up to 1 microM but attenuated (+)8-hydroxy-2-(di-n-propylamino) tetralin-induced inhibition of cell firing with an apparent pKb value of 9.5. SB-649915 (1 microM) significantly attenuated exogenous 5-HT-induced inhibition of electrically-stimulated [3H]5-HT release from guinea pig cortex. In studies designed to enhance endogenous 5-HT levels, and therefore increase tone at 5-HT1B autoreceptors, SB-649915 significantly potentiated [3H]5-HT release at 100 and 1000 nM. In LLCPK cells expressing human recombinant 5-HT transporters and in rat cortical synaptosomes, SB-649915 inhibited [3H]5-HT re-uptake with pIC50 values of 7.9 and 9.7, respectively. In summary, SB-649915 is a novel, potent 5-HT1A/1B autoreceptor antagonist and 5-HT re-uptake inhibitor in native tissue systems and represents a novel mechanism that could offer fast acting antidepressant action.     

Differential regulation of rat peripheral 5-HT<Subscript>2A</Subscript> and 5-HT<Subscript>2B</Subscript> receptor systems: influence of drug treatment

Most studies of 5-HT₂ receptor regulation have been carried out on the central nervous system (CNS) (which expresses 5-HT_2A and 5-HT_2C receptors); very few in vitro studies have addressed the peripheral receptors 5-HT_2A and 5-HT_2B. The aim of this investigation was to compare the possible short- and long-term processes regulating these peripheral receptors in the rat.The in vitro contractile response elicited by serotonin (5-HT, 10 µM) in the rat gastric fundus (5-HT_2B receptor system) was rapid and followed by a partial fade to a steady state, in contrast with the rat thoracic aorta response (5-HT_2A receptor system), which was more stable, slower and sustained. To characterize drug-receptor interactions, cumulative concentration/response curves (CCRCs) for 5-HT were constructed ex vivo for rat tissues treated with drugs acting at these receptors. Rats were examined 4 or 24 h after a single, i.p. administration of (±)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane [(±)DOI, 1 or 2.5 mg/kg], clozapine, cyproheptadine or rauwolscine (10 mg/kg), 48 h after a single i.p. administration of (±)DOI (2.5 mg/kg), clozapine or cyproheptadine (10 mg/kg) or 24 h after the last of with 15 daily i.p. administrations of (±)DOI (1 or 2.5 mg/kg), clozapine, cyproheptadine or rauwolscine (10 mg/kg). In the aorta, E_max (the maximum response elicited by 5-HT) was unchanged 4 h after a single dose of any of the drugs tested. However, 24 h after a single dose, E_max was lower in animals treated with (±)DOI (2.5 mg/kg), clozapine or cyproheptadine than in controls, whilst 48 h after a single dose of (±)DOI (2.5 mg/kg), clozapine or cyproheptadine there was no difference in E_max between experimental and control animals. After chronic treatment with (±)DOI (2.5 mg/kg), clozapine and cyproheptadine, E_max was lower than in controls. In the gastric fundus, E_max 4 h after a single dose of each drug was lower than in controls, and the response recovered by 24 or 48 h. Following chronic treatment, E_max was significantly lower than in controls for each drug used.These findings suggest first, that regulation of peripheral 5-HT₂ receptors (5-HT_2A and 5-HT_2B) is a functionally significant phenomenon in vivo, and occurs after administration of both agonists and antagonists. Second, the kinetics of peripheral 5-HT₂ receptor regulation were similar in both in vivo and ex vivo experiments. The 5-HT_2B receptors in rat gastric fundus are more sensitive to drug-induced regulation than the 5-HT_2A rat aortic receptors. Finally, long-term regulation of both receptors stabilizes short-term desensitization for longer.     

Synergism between 5-HT1B/1D and 5-HT1A receptor antagonists on turnover and release of 5-HT in guinea-pig brain in vivo

The effects on 5-HT turnover (5-HIAA/5-HT ratio) and extracellular 5-HT and 5-HIAA levels (in vivo microdialysis in freely moving animals) were analysed in guinea-pig brains following the 5-HT_1B receptor antagonist, GR 127935 {N-[4-methoxy-3-(4-methyl-1-piper_{azinyl)phenyl]-2’-methyl-4’-(5-methyl-1,2,4-oxadiazol-3-}yl) [1,1-biphenyl]-4-carboxamide}, or the 5-HT_1A receptor antagonist, WAY-100635 (N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride), administered alone or in combination. GR 127935, injected alone, increased 5-HT turnover with maximal effects approximately 50% above the control levels in the four brain regions examined (hypothalamus, hippocampus, striatum and frontal cortex). GR 127935 significantly increased extracellular concentrations of 5-HT and 5-HIAA in frontal cortex (40%), whereas 5-HIAA, but not 5-HT, was elevated in striatum (20–30%). WAY-100635 did not significantly change 5-HT turnover but caused a small significant increase in the extracellular 5-HT and 5-HIAA concentrations in both striatum and frontal cortex. The combined treatment with GR 127935 and WAY-100635 resulted in an increased 5-HT turnover reaching maximal effects of 70–90% above the control values in all brain regions tested and produced a significant elevation of striatal and frontal cortex extracellular 5-HT (40% and 60%, respectively) and 5-HIAA (60% and 70%, respectively) concentrations. The synergistic effect of the two receptor antagonists on the 5-HT turnover and the terminal release of 5-HT indicate somatodendritic 5-HT release and stimulation of inhibitory 5-HT_1A receptors at this level. Extracellular 5-HIAA seems to be a better marker than 5-HT itself for the evoked 5-HT release when the reuptake mechanism is intact. Received: 2 September 1998 / Accepted: 19 November 1998     

Further characterization of the 5-HT<Subscript>1</Subscript> receptors mediating cardiac sympatho-inhibition in pithed rats: pharmacological correlation with the 5-HT<Subscript>1B</Subscript> and 5-HT<Subscript>1D</Subscript> subtypes

Serotonin (5-hydroxytryptamine; 5-HT) is capable of inhibiting the tachycardic responses elicited by sympathetic stimulation, but not by exogenous noradrenaline, in pithed rats pre-treated with desipramine. More recently, it has been shown that this cardiac sympatho-inhibitory response to 5-HT, mediated by prejunctional 5-HT₁ receptors as well as putative 5-ht_5A/5B receptors, is mimicked dose-dependently by the agonists CP 93,129 (r5-HT_1B), sumatriptan (5-HT_1B/1D) and PNU-142633 (5-HT_1D). This study analysed further the pharmacological profile of the above 5-HT₁ receptors.Continuous i.v. infusions of CP 93,129, sumatriptan or PNU-142633 (30 µg kg^–1min^–1 each) failed to modify the tachycardic responses to exogenous noradrenaline but inhibited those elicited by preganglionic (C7–T1) stimulation of the cardiac sympathetic outflow. These sympatho-inhibitory responses were unaltered after i.v. administration of physiological saline (1 ml kg^–1) or the 5-HT_1A receptor antagonist WAY 100635 (10 µg kg^–1). In contrast, the antagonist GR 127935 (5-HT_1B/1D; 100 µg kg^–1, i.v.) abolished the responses to CP 93,129, sumatriptan and PNU-142633, whilst SB224289 (5-HT_1B; 300 µg kg^–1, i.v.) abolished the responses to CP 93,129 without affecting those to sumatriptan and PNU-142633. Interestingly, BRL15572 (5-HT_1D; 300 µg kg^–1, i.v.) abolished the responses to PNU-142633 and attenuated those to sumatriptan, but not those to CP 93,129.WAY 100635, GR 127935, SB224289 and BRL15572, given alone at the above doses, failed to modify the sympathetically induced tachycardic responses. The 5-HT₁ receptors producing cardiac sympatho-inhibition in pithed rats thus display the pharmacological profile of the 5-HT_1B and 5-HT_1D receptor subtypes.     

Acute and chronic tryptophan depletion differentially regulate central 5-HT1A and 5-HT2A receptor binding in the rat

Rationale Tryptophan depletion is used to reduce central serotonergic function and to investigate its role in psychiatric illness. Despite widespread clinical use, its effects on serotonin (5-HT) receptors have not been well characterized. Objective The aim of this study was to examine the effect of acute (ATD) and chronic tryptophan depletion (CTD) on free-plasma tryptophan (TRP), central TRP and 5-HT and brain 5-HT_1A and 5-HT_2A receptor binding in the rat. Methods TRP and 5-HT were measured by high-performance liquid chromatography and receptor levels determined by homogenate radioligand binding and in-vitro receptor autoradiography. Results Free-plasma TRP, central TRP and central 5-HT levels were significantly and similarly reduced by ATD and 1- and 3-week CTD compared to controls. ATD significantly reduced 5-HT_1A binding in the dorsal raphe (14%) but did not significantly alter postsynaptic 5-HT_1A binding (frontal cortex, remaining cortex and hippocampus) or 5-HT_2A binding (cortex and striatum). One-week CTD did not significantly alter cortical 5-HT_2A binding or postsynaptic 5-HT_1A binding. Furthermore, 3-week CTD did not significantly alter 5-HT_1A binding but significantly increased cortical 5-HT_2A binding without affecting striatal or hippocampal levels. In the CTD 1 and 3-week groups, rat body weight was significantly decreased as compared to controls. However, weight loss was not a confounding factor for decreased cortical 5-HT_2A-receptor binding. Conclusion ATD-induced reduction in somatodendritic 5-HT_1A autoreceptor binding may represent an intrinsic ‘homeostatic response’ reducing serotonergic feedback in dorsal raphe projection areas. In contrast, the increase in 5-HT_2A receptor after CTD may be a compensatory response to a long-term reduction in 5-HT.     

Pharmacological characterisation of the agonist radioligand binding site of 5-HT<Subscript>2A</Subscript>, 5-HT<Subscript>2B</Subscript> and 5-HT<Subscript>2C</Subscript> receptors

In the present study we compared the affinity of various drugs for the high affinity agonist-preferring binding site of human recombinant 5-HT_2A, 5-HT_2B and 5-HT_2C receptors stably expressed in monoclonal mammalian cell lines. To ensure that the agonist-preferring conformation of the receptor was preferentially labelled in competition binding experiments, saturation analysis was conducted using antagonist and agonist radiolabels at each receptor. Antagonist radiolabels ([³H]-ketanserin for 5-HT_2A receptor and [³H]-mesulergine for 5-HT_2B and 5-HT_2C receptor) bound to a larger population of receptors in each preparation than the corresponding agonist radiolabel ([¹²⁵I]-DOI for 5-HT_2A receptor binding and [³H]-5-HT for 5-HT_2B and 5-HT_2C receptor binding). Competition experiments were subsequently conducted against appropriate concentrations of the agonist radiolabels bound to the agonist-preferring subset of receptors in each preparation. These studies confirmed that there are a number of highly selective antagonists available to investigate 5-HT₂ receptor subtype function (for example, MDL 100907, RS-127445 and RS-102221 for 5-HT_2A, 5-HT_2B and 5-HT_2C receptors respectively). There remains, however, a lack of highly selective agonists. (–)DOI is potent and moderately selective for 5-HT_2A receptors, BW723C86 has poor selectivity for human 5-HT_2B receptors, while Org 37684 and VER-3323 display some selectivity for the 5-HT_2C receptor. We report for the first time in a single study, the selectivity of numerous serotonergic drugs for 5-HT₂ receptors from the same species, in mammalian cell lines and using, exclusively, agonist radiolabels. The results indicate the importance of defining the selectivity of pharmacological tools, which may have been over-estimated in the past, and highlights the need to find more selective agonists to investigate 5-HT₂ receptor pharmacology.     

Destruction of serotonergic nerve terminals prevents fluoxetine-induced desensitization of hypothalamic 5-HT<Subscript>1A</Subscript> receptors

Abstract Rationale. Selective serotonin (5-HT, 5-hydroxytryptamine) reuptake inhibitors (SSRIs) such as fluoxetine produce a gradual desensitization of hypothalamic post-synaptic 5-HT_1A receptor systems. It is assumed that the effects of SSRIs on post-synaptic 5-HT_1A receptors are mediated by 5-HT reuptake inhibition, leading to an increase of 5-HT in the synapse. However, there is no direct evidence to support this hypothesis. Objectives. The present study determined whether 5-HT_1A receptor desensitization was mediated by fluoxetine's effects on serotonergic nerve terminals. Methods. Serotonergic nerve terminals were destroyed by intracerebroventricular (i.c.v.) injection of the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) combined with injection of the norepinephrine/dopamine reuptake inhibitor nomifensine. 5,7-DHT-induced loss of serotonergic terminals was confirmed by a 95% reduction in the density of [³H]paroxetine-labeled 5-HT transporters in the hypothalamus and a 97% reduction in the levels of 5-HT and 5-hydroxyindoleacetic acid in the cortex. Two weeks after the 5,7-DHT injections, rats were injected daily with fluoxetine (5 mg/kg or 10 mg/kg, i.p.) or saline for 14 days. Results. Injections of 10 mg/kg fluoxetine produced a significant decrease in body weight gain. Destruction of serotonergic nerve terminals reduced body weight and potentiated the ability of fluoxetine to further inhibit body weight gain. Increases in plasma levels of adrenal corticotrophic hormone (ACTH, corticotropin), corticosterone, and oxytocin after injection of the 5-HT_1A agonist 8-hydroxy-2-dipropylaminotetralin [(±)8-OH-DPAT] were used as peripheral markers of 5-HT_1A receptor function in the hypothalamus. In vehicle-pretreated rats, fluoxetine produced a dose-dependent reduction in the (±)8-OH-DPAT-induced increase in plasma ACTH and oxytocin levels. Destruction of serotonergic nerve terminals blocked the ability of fluoxetine to produce a desensitization in the ACTH, corticosterone, and oxytocin responses to (±)8-OH-DPAT. Conclusions. The ability of fluoxetine to induce a desensitization of hypothalamic post-synaptic 5-HT_1A receptor systems is dependent on the integrity of serotonergic nerve terminals in the hypothalamus, while its effect on body weight is not mediated by serotonergic nerve terminals in the hypothalamus. Electronic Publication     

Intraseptal injection of the 5-HT<Subscript>1A</Subscript>/5-HT<Subscript>7</Subscript> agonist 8-OH-DPAT and working memory in rats

Rationale In rats, 5-HT_1A receptors are present in the septal region, e.g. on cholinergic neurons of the medial septum, where they might be a substrate for cognitively relevant interactions between cholinergic and serotonergic systems.Objective The present experiment assessed the effects of the stimulation of septal 5-HT_1A receptors on spatial working memory.Methods Stimulation of septal 5-HT_1A receptors was carried out by infusions targetting the medial septum of the 5-HT_1A/5-HT₇ receptor agonist 8-hydroxy-2-(di-n-propyl-amino)-tetralin (8-OH-DPAT; 0.5 or 4 µg). Spatial memory was assessed in a water maze using a protocol placing emphasis on spatial working memory. The location of the hidden platform was changed every day and performance was assessed on two consecutive trials each day.Results In comparison to vehicle injections, the intraseptal infusion of 4 µg 8-OH-DPAT impaired performance significantly: rats treated with 8-OH-DPAT exhibited increased distances to reach the hidden platform on both trials 1 and 2. Rats infused with 0.5 µg showed similar changes that failed to be significant. Such effects were not observed when the platform was visible.Conclusions These results extend those of a previous experiment which showed that intraseptal injections of 8-OH-DPAT impaired spatial reference memory. Based on the characteristics of the observed deficits, it is suggested that the 8-OH-DPAT-induced impairment, rather than being only the result of a true alteration of working memory, might reflect a more global cognitive deficiency in which alteration of general memory capacities may be biased by disrupted search strategies/exploration and/or dysfunctions of attention.H. Jeltsch and F. Bertrand contributed equally to the work.     

SB-649915-B, a novel 5-HT1A/B autoreceptor antagonist and serotonin reuptake inhibitor, is anxiolytic and displays fast onset activity in the rat high light social interaction test.

Preclinically, the combination of an SSRI and 5-HT autoreceptor antagonist has been shown to reduce the time to onset of anxiolytic activity compared to an SSRI alone. In accordance with this, clinical data suggest the coadministration of an SSRI and (+/-) pindolol can decrease the time to onset of anxiolytic/antidepressant activity. Thus, the dual-acting novel SSRI and 5-HT(1A/B) receptor antagonist, SB-649915-B, has been assessed in acute and chronic preclinical models of anxiolysis. SB-649915-B (0.1-1.0 mg/kg, i.p.) significantly reduced ultrasonic vocalization in male rat pups separated from their mothers (ED(50) of 0.17 mg/kg). In the marmoset human threat test SB-649915-B (3.0 and 10 mg/kg, s.c.) significantly reduced the number of postures with no effect on locomotion. In the rat high light social interaction (SI), SB-649915-B (1.0-7.5 mg/kg, t.i.d.) and paroxetine (3.0 mg/kg, once daily) were orally administered for 4, 7, and 21 days. Ex vivo inhibition of [(3)H]5-HT uptake was also measured following SI. SB-649915-B and paroxetine had no effect on SI after 4 days. In contrast to paroxetine, SB-649915-B (1.0 and 3.0 mg/kg, p.o., t.i.d.) significantly (p<0.05) increased SI time with no effect on locomotion, indicative of an anxiolytic-like profile on day 7. Anxiolysis was maintained after chronic (21 days) administration by which time paroxetine also increased SI significantly. 5-HT uptake was inhibited by SB-649915-B at all time points to a similar magnitude as that seen with paroxetine. In conclusion, SB-649915-B is acutely anxiolytic and reduces the latency to onset of anxiolytic behavior compared to paroxetine in the SI model.     

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT_1B receptor ligand, and of BRL-15572, a preferential h5-HT_1D receptor ligand, on the presynaptic h5-HT_1B and h5-HT_1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT_1D heteroreceptors in the human atrium. The brain preparations, preincubated with [³H]serotonin ([³H]5-HT), and the segments of atrial appendages, preincubated with [³H]noradrenaline, were superfused with modified Krebs’ solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K⁺ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2μM; concentration 154 times higher than its K_i at h5-HT_1D receptors) but was antagonized by SB-216641 (0.1μM; concentration 100 times higher than its K_i at h5-HT_1B receptors; apparent pA₂ 8.45). SB-216641 (0.1μM) by itself facilitated, whereas BRL-15572 (2μM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1μM) also facilitated, and BRL-15572 (2μM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K⁺-evoked tritium overflow. This response was unaffected by BRL-15572 (300nM) but antagonized by SB-216641 (15nM; drug concentrations 23 and 15 times higher than their K_i at h5-HT_1D and h5-HT_1B receptors, respectively). Both drugs, given alone, did not modify the K⁺-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300nM; 23 times K_i at h5-HT_1D receptors) but not by SB-216641 (30nM; 30 times K_i at h5-HT_1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT_1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT_1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT_1B and 5-HT_1D receptors in functional studies. Received: 14 March 1997 / Accepted: 18 May 1997     

Pharmacological characterization of mitogen-activated protein kinase activation by recombinant human 5-HT<Subscript>2C</Subscript>, 5-HT<Subscript>2A</Subscript>, and 5-HT<Subscript>2B</Subscript> receptors

The type 2 serotonin (5-HT₂) receptor subfamily is known to couple to phosphoinositide hydrolysis (PI) and the subsequent mobilization of intracellular Ca²⁺, as well as the release of arachidonic acid (AA). Less is known of 5-HT₂-mediated activation of the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK1/2) signaling. The present study measured the relative efficacies and potencies of 5-HT agonists to activate ERK2 in non-neuronal cells expressing recombinant human 5-HT_2A, 5-HT_2B, and 5-HT_2C(ISV) receptors. 5-HT agonists stimulated ERK2 activity via all three 5-HT₂ subtypes. There were no meaningful differences in the potencies or relative efficacies of these agonists to affect ERK2 activity vs. PI accumulation or Ca²⁺ mobilization, suggesting that these pathways may be sequentially linked. Indeed, ERK2 activity was very sensitive to PKC inhibition and calcium chelation and insensitive to tyrosine kinase and PI-3-kinase inhibition. 5-HT₂ receptors efficiently couple to MAPK activation via sequential PI hydrolysis, and Ca²⁺ mobilization. This profile differs from reports of “agonist-directed trafficking of receptor stimulus” between PI/Ca²⁺ and AA pathways activated by 5-HT₂ receptors.     

Effect of chronic administration of the selective serotonin (5-HT) uptake inhibitor citalopram on extracellular 5-HT and apparent autoreceptor sensitivity in rat forebrain in vivo

Rats were administered the selective serotonin (5-HT) uptake blocker citalopram or saline for 14 days to determine if prolonged treatment would lead to changes in extracellular 5-HT or autoreceptor sensitivity. One day after drug withdrawal, dialysis probes were implanted in the frontal cortex and dorsal hippocampus. Dialysis experiments were carried out using chloral hydrate anesthetized rats. The experimental protocol comprised the administration of three consecutive drug challenges: (1) After stable baseline levels were obtained, citalopram was infused through the dialysis probes to locally block uptake in the forebrain. (2) Subsequently, a 5-HT_1B receptor agonist (RU24969 or CP93,129) was infused through the probe to test for changes in terminal autoreceptor sensitivity. (3) Last, citalopram was administered systemically to test the effect of indirect activation of somatodendritic autoreceptors. Under these conditions, with uptake already blocked locally in the forebrain, systemic citalopram produces a decrease in extracellular 5-HT, an effect that can be inhibited by pretreatment with antagonists of 5-HT_1A receptors. The results indicate that during local infusion of citalopram extracellular 5-HT was significantly higher in the dorsal hippocampus of the chronic citalopram as compared to saline treatment group. This difference persisted throughout the full time course of the experiment. However, the decreases in 5-HT levels produced by local infusion of a 5-HT_1B receptor agonist or after systemic citalopram administration were not significantly different between the chronic citalopram and saline treated groups. There were no significant differences between chronic citalopram and saline treated animals in frontal cortex. These results suggest that prolonged inhibition of 5-HT uptake may produce a selective change in the regulation of release from median raphe 5-HT neurons, but this change could not be clearly linked to a change in nerve terminal or somatodendritic autoreceptor sensitivity.     

5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression

1. In the human temporal artery both 5-HT_1-like and 5-HT_2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT_1-like receptors resemble more closely recombinant 5-HT_1B than 5-HT_1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT_1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2-methyl-4′[(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-yl] carbonyl} furo[2,3-f]indole-3-spiro-4′-piperidine oxalate) and the 5-HT_1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT_1B, 5-HT_1D and other 5-HT receptors.
2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent K_B=1 nM) and ketanserin (1 μM) but not by BRL-15572 (500 nM).
3. Sumatriptan evoked contractions (EC₅₀, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM).
4. The potency of 5-HT (EC₅₀) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20–0.67, the remainder being mediated through ketanserin-sensitive receptors.
5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT_1B (8/8), 5-HT_1D (2/8), 5-HT_1F (0/4), 5-HT_2A (0/8), 5-HT_2B (0/8), 5-HT_2C (0/8), 5-HT₄ (4/8) and 5-HT₇ (4/8).
6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT_1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT_1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.

     

Effect of 5-HT<Subscript>3</Subscript> antagonists and a 5-HT<Subscript>1A</Subscript> agonist on fluoxetine-induced conditioned gaping reactions in rats

Rationale  The effect of manipulation of the serotonin (5-HT) system on conditioned gaping (presumably reflective of nausea in rats) was evaluated. Objective  The potential of the selective serotonin reuptake inhibitor (SSRI), fluoxetine (which produces nausea in the clinic), to produce conditioned gaping in rats and of the 5-HT₃ antagonists (ondansetron and palonosetron) and the 5-HT_1A autoreceptor agonist (8-OH-DPAT) to reverse this effect were evaluated. Materials and methods  In each of four experiments, rats received three pairings of intraorally delivered 17% sucrose solution and fluoxetine (0, 2, 10 or 20 mg/kg) and 72 h later were given a drug-free test trial. In experiment 2, rats were pretreated with the 5-HT₃ antagonists, ondansetron (0, 0.1 or 1.0 mg/kg) or the longer acting palonosetron (0.1 mg/kg), 30 min before each of three sucrose–fluoxetine (20 mg/kg) pairings. In experiment 3, rats were injected with palonosetron (0.1 mg/kg) 2 h before each of three sucrose–fluoxetine (20 mg/kg) or sucrose–lithium chloride (LiCl, 25 mg/kg) pairings. In experiment 4, rats were pretreated with the 5-HT_1A autoreceptor agonist, 8-OH-DPAT (DPAT, 0.1 mg/kg) 30 min before each of three sucrose–fluoxetine (20 mg/kg) pairings. Results  After two sucrose–fluoxetine pairings, the highest dose of fluoxetine tested (20 mg/kg) produced conditioned gaping reactions. These conditioned gaping reactions were prevented by pretreatment with DPAT, but not with the 5-HT₃ antagonists. On the other hand, palonosetron administered 2 h prior to sucrose–LiCl pairings attenuated conditioned gaping reactions. Conclusions  These results suggest that the conditioned nausea produced by SSRIs, but not LiCl, may be resistant to treatment with 5-HT₃ antagonists, but not 5-HT_1A autoreceptor agonists.