The 5-HT<Subscript>1A</Subscript> agonist 8-OH-DPAT dose-dependently interferes with the establishment and the expression of lithium-induced conditioned rejection reactions in rats

Abstract Rationale. The present experiments evaluated the potential of the 5-HT_1A agonist, 8-OH-DPAT (DPAT), which reduces serotonin availability, to interfere with both the establishment and with the expression of lithium-induced conditioned rejection reactions (experiment 1) and lithium-induced taste avoidance (experiment 2). Objectives. To determine the effect of reduced serotonin availability on conditioned rejection reactions, a rat model of nausea. Methods. Rats were injected with 8-OH-DPAT [at doses of 0.0 (saline), 0.01 or 0.1 mg/kg, SC] 30 min prior to exposure to 0.1% saccharin solution by intra-oral infusion (experiment 1) or by bottle presentation (experiment 2). Immediately following saccharin exposure, rats were injected with 20 ml/kg lithium chloride (0.15 M) or 20 ml/kg saline solution. On each of three test trials, rats were injected with DPAT [0.0 (saline), 0.01 and 0.1 mg/kg, SC; counterbalanced order], 30 min prior to exposure to saccharin solution by intra-oral infusion (experiment 1) or by a two-bottle test (experiment 2: saccharin and water). Results. DPAT interfered with both the establishment (at a dose of 0.1 mg/kg, SC) and with the expression (at doses of 0.01 and 0.1 mg/kg, SC) of lithium-induced conditioned rejection reactions; however, DPAT did not modulate taste avoidance in a consumption test. Conclusions. These results indicate that conditioned rejection reactions, but not taste avoidance, can be attenuated by the anti-emetic agent, 8-OH-DPAT. 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.     

A PET study on regional coexpression of 5-HT<Subscript>1A</Subscript> receptors and 5-HTT in the human brain

Rationale Several lines of evidence suggest inter-dependency between the serotonin transporter (5-HTT) and the 5HT_1A receptor, two recognised targets for the treatment of anxiety and depression. Objectives to examine the correlation of regional expression levels for these two serotonergic markers in the human brain in vivo. Methods Twelve male control subjects were examined with PET twice on the same day, using the radioligands [¹¹C]WAY 100635 and [¹¹C]MADAM for quantification of the 5-HT_1A receptor and the 5-HTT, respectively. The binding potential (BP) was calculated for raphe nuclei, hippocampus and frontal cortex. Results In all regions, the BP for both [¹¹C]WAY 100635 (raphe nuclei 1.85–4.71, hippocampus 2.52–6.17, frontal cortex 2.03–3.79) and [¹¹C]MADAM (2.70–7.65, 0.47–1.76, 0.18–0.51) varied several fold between subjects. In the raphe nuclei, where the two markers are situated on the same neurons, the ratio of [¹¹C]WAY 100635 binding to [¹¹C]MADAM BP binding varied considerably (0.43–1.05). There was a positive correlation between the two markers in the raphe nuclei (r _xy = 0.68, p < 0.05) and in the hippocampus (r _xy = 0.97, p < 0.001) but not in the frontal cortex (r _xy = −0.25, p = 0.44). Conclusions The results support a correlation between density levels of the 5-HT_1A-receptor and the 5-HTT in the raphe nuclei and hippocampus but not in the frontal cortex. A suggested clinical implication is that the inter-individual variability in 5-HT_1A-receptor and 5-HTT densities, as well as the ratio of these, is of particular interest in relation to individual responses to selective serotonin reuptake inhibitor treatment.     

Chromium treatment decreases the sensitivity of 5-HT<Subscript>2A</Subscript> receptors

RATIONALE: Recent case series suggest that chromium picolinate in doses of 400 microg daily may have antidepressant properties, perhaps through increasing the peripheral availability of tryptophan for brain serotonin (5-HT) synthesis. OBJECTIVES: To determine the effects of chromium treatment on plasma tryptophan availability and on brain 5-HT function in human and animal models. METHODS: We studied the effects of short-term chromium supplementation on plasma concentrations of tryptophan and other large neutral amino acids. Brain 5-HT function was assessed by measuring the corticosterone/cortisol response to the 5-HT precursor, 5-hydroxytryptophan (5-HTP), a response believed to be mediated via indirect activation of 5-HT(2A) receptors. RESULTS: In rats, chromium increased peripheral and central tryptophan availability and elevated brain 5-HT content. Changes in peripheral tryptophan availability were not seen in humans but in both rats and humans, chromium lowered the cortisol response to challenge with 5-HTP. CONCLUSIONS: Chromium can modify brain 5-HT function in humans and animals, perhaps by altering the sensitivity of central 5-HT(2A) receptors.     

Paroxetine is effective in desensitizing 5-HT<Subscript>1A</Subscript> receptor function in adult offspring exposed prenatally to cocaine

Rationale Desensitization of postsynaptic 5-HT_1A receptors may be responsible for the therapeutic effectiveness of serotonin selective uptake inhibitors (SSRIs). As prenatal cocaine exposure produces long-term deficits in 5-HT neurons in offspring, it may alter the ability of postsynaptic 5-HT_1A receptors to be desensitized by chronic paroxetine.Objectives The aim of the study is to determine (1) prenatal cocaine-induced changes in 5-HT_1A receptor function and (2) the effectiveness of chronic treatment with paroxetine to produce 5-HT_1A receptor desensitization in adult offspring exposed to cocaine in utero.Methods Pregnant rats received saline or (–)cocaine (15 mg/kg, s.c.) twice daily from gestational days 13 through 20. Adult male offspring from each of prenatal groups were treated with saline or paroxetine (10 mg/kg/day; i.p.) for 14 days. Eighteen hours post-treatment, rats were challenged with saline or the 5-HT_1A receptor agonist (+)8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.04 or 0.2 mg/kg, s.c.). Plasma oxytocin, adrenocorticotropic hormone (ACTH), corticosterone, renin and prolactin were determined.Results Prenatal cocaine exposure did not alter 5-HT_1A receptor-mediated neuroendocrine responses. Paroxetine treatment desensitized 5-HT_1A receptor-mediated increases in oxytocin, ACTH and corticosterone to a comparable extent in all offspring and reduced the E_max for ACTH only in prenatal cocaine-exposed offspring. Cortical [³H]-8-OH-DPAT- or [³H]-WAY100635-labeled 5-HT_1A receptors were unaltered by prenatal cocaine or subsequent paroxetine treatment.Conclusions Postsynaptic 5-HT_1A receptor function is unaltered by prenatal cocaine exposure and paroxetine can effectively desensitize 5-HT_1A receptor function in adult cocaine-exposed offspring. These data suggest that paroxetine may be clinically effective in treating mood disorders in adults exposed in utero to cocaine.This study was supported by United States Public Health Services grants DA07741 (G.B.) and DA13669 (L.VDK.)     

5-HT<Subscript>1A</Subscript> agonists: alcohol drinking in rats and squirrel monkeys

Rationale. Increased alcohol intake after administration of low doses of 5-HT_1A agonists is thought to be due to a reduction in 5-HT impulse flow due to activation of 5-HT_1A somatodendritic receptors, whereas decreased alcohol drinking found after administration of higher doses of 5-HT_1A agonists may be mediated by action at postsynaptic 5-HT_1A receptors. Objective. This study compares Long-Evans rats and squirrel monkeys to examine the hypothesis that low doses of the 5-HT_1A selective agonists, 8-OH-DPAT and alnespirone, will preferentially increase, and at higher doses decrease alcohol drinking, and whether these effects can be antagonized by WAY 100635. Methods. Male Long-Evans rats were induced to drink from two bottles, one containing a solution of 10% ethanol and 1% sucrose (w/v), the other containing an equally preferred concentration of sucrose. Squirrel monkeys also drank from two bottles, one containing a solution of 2% ethanol and 15% sucrose (w/v), the other, water. Results. In rats, low doses of both 8-OH-DPAT (0.018–0.03 mg/kg) and alnespirone (0.3–3.0 mg/kg) increased alcohol drinking by ca. 100% without altering sucrose intake. The highest dose of 8-OH-DPAT (0.1 mg/kg) suppressed intake of both solutions without significant motor impairment. Pretreatment with WAY 100635 (0.1 mg/kg), shifted the entire dose-effect curve of 8-OH-DPAT to the right, and antagonized the effects of the 0.56 mg/kg dose of alnespirone. In the monkeys, administration of both agonists dose-dependently decreased alcohol intake and were behaviorally sedative. Conclusions. These results support the hypothesis that in rats, 5-HT_1A receptor stimulation activates somatodendritic receptors at lower doses and postsynaptic receptors at higher doses, each with opposite effects on alcohol intake. The absence of such biphasic dose-effect curves in monkeys suggests a different function of 5-HT_1A somatodendritic receptors in rats and monkeys, at least with regard to alcohol drinking. Electronic Publication     

A positron emission tomography (PET) study of cerebral dopamine D<Subscript>2</Subscript> and serotonine 5-HT<Subscript>2A</Subscript> receptor occupancy in patients treated with cyamemazine (Tercian)

Rationale Cyamemazine (Tercian) is an antipsychotic drug with anxiolytic properties. Recently, an in vitro study showed that cyamemazine possesses high affinity for serotonin 5-HT_2A receptors, which was fourfold higher than its affinity for dopamine D₂ receptors (Hameg et al. 2003).Objectives The aim of this study is to confirm these previous data in vivo in patients treated with clinically relevant doses of Tercian.Methods Eight patients received 37.5, 75, 150 or 300 mg/day of Tercian depending on their symptomatology. Dopamine D₂ and serotonin 5-HT_2A receptor occupancies (RO) were assessed at steady-state plasma levels of cyamemazine with positron emission tomography (PET), using [¹¹C]raclopride and [¹¹C]N-methyl-spiperone, respectively. The effective plasma level of the drug leading to 50% of receptor occupancy was estimated by fitting RO with plasma levels of cyamemazine at the time of the PET scan.Results Cyamemazine induced near saturation of 5-HT_2A receptors (RO=62.1–98.2%) in the frontal cortex even at low plasma levels of the drug. On the contrary, occupancy of striatal D₂ receptors increased with plasma levels, and no saturation was obtained even at high plasma levels (RO=25.2–74.9%). The effective plasma level of cyamemazine leading to 50% of D₂ receptor occupancy was fourfold higher than that for 5-HT_2A receptors. Accordingly, individual 5-HT_2A/D₂ RO ratios ranged from 1.26 to 2.68. No patients presented relevant increased prolactin levels, and only mild extrapyramidal side effects were noticed on Simpson and Angus Scale.Conclusion This in vivo binding study conducted in patients confirms previous in vitro findings indicating that cyamemazine has a higher affinity for serotonin 5-HT_2A receptors compared to dopamine D₂ receptors. In the dose range 37.5–300 mg, levels of dopamine D₂ occupancy remained below the level for motor side effects observed with typical antipsychotics and is likely to explain the low propensity of the drug to induce extrapyramidal side effects.     

Occupancy of 5-HT<Subscript>1A</Subscript> receptors by clozapine in the primate brain: a PET study

Abstract Rationale. The pharmacological mechanism underlying the atypical properties of the antipsychotic drug clozapine remains to be identified. The serotonin 5-hydroxytryptamine-1A (5-HT_1A) receptor subtype has been suggested to play a role in the pathophysiology of schizophrenia and is one among several central neuroreceptors for which clozapine has moderate affinity in vitro. Objective. The aim of this positron emission tomography (PET) study was to determine 5-HT_1A receptor occupancy in the monkey brain after IV injection of clozapine in doses that previously have been shown to give plasma concentrations representative of the 200 to 800 mg oral dose range recommended for clinical management of patients. Methods. Each of four cynomolgus monkeys was examined three times on the same day with PET and the radioligand [carbonyl-¹¹C]WAY-100635. The first measurement was performed at baseline conditions, the second after clozapine 1.5 mg/kg and the third after 6 mg/kg. Two additional monkeys were examined at baseline and after 15 mg/kg IV. Central 5-HT_1A receptor occupancy was calculated using an equilibrium-ratio analysis. Results. The occupancy ranged from 23 to 34% after 1.5 mg/kg clozapine and from 36 to 49% after 6 mg/kg in different brain regions of the four monkeys. The regional receptor occupancy values after 15 mg/kg were between 39 and 51% in the two monkeys. There was no evident difference between the frontal cortex, the temporal cortex and the raphe nucleus. Conclusion. The study shows that clozapine occupies 5-HT_1A receptors in the primate brain at clinically representative plasma concentrations. The results support that the 5-HT_1A receptor is a candidate target for the atypical drug actions of clozapine. Electronic Publication     

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.     

EEG effects of buspirone and pindolol: a method of examining 5-HT<Subscript>1A</Subscript> receptor function in humans

Rationale. An involvement of 5-HT_1A receptors is postulated in the pathophysiology of affective disorders and mechanism of action of antidepressants. Methods for studying their functional integrity in humans are, however, limited. Preliminary data suggests that activation of somatodendritic 5-HT_1A receptors cause a negative shift in the EEG frequency spectrum. Animal research suggests that pindolol is an agonist at these receptors but an antagonist at postsynaptic 5-HT_1A receptors. Objective. We postulated that while pindolol would antagonise known postsynaptic mediated neuroendocrine responses to the 5-HT_1A agonist buspirone, both drugs would have a similar effect on the EEG frequency spectrum. Methods. Fourteen healthy men were administered placebo or pindolol (20 mg orally) 90 min before placebo or buspirone (30 mg orally) in a double blind cross-over study. Plasma prolactin and growth hormone were assayed and EEGs recorded before and after drug administration. Results. A significant negative shift in the EEG frequency spectrum was found for both buspirone and pindolol, with the combination producing a similar effect to each drug alone. In contrast, the neuroendocrine response to buspirone was significantly attenuated by pindolol. Conclusions. The data obtained are consistent with the EEG effects of buspirone and pindolol being mediated by somatodendritic 5-HT_1A receptors, in contrast to the neuroendocrine response, which is known to be mediated by postsynaptic receptors. The development of this novel method of assessing somatodendritic 5-HT_1A receptors in humans is a potentially important advance which may allow the testing of hypotheses of its involvement in depression and response to antidepressants. Electronic Publication     

Comparison of hippocampal G protein activation by 5-HT<Subscript>1A</Subscript> receptor agonists and the atypical antipsychotics clozapine and S16924

This study employed [³⁵S]guanosine 5-O-(3-thiotriphosphate) ([³⁵S]GTPS) binding to compare the actions of antipsychotic agents known to stimulate cloned, human 5-HT_1A receptors with those of reference agonists at postsynaptic 5-HT_1A receptors. In rat hippocampal membranes, the following order of efficacy was observed (maximum efficacy, E_max, values relative to 5-HT=100): (+)8-OH-DPAT (85), flesinoxan (62), eltoprazine (60), S14506 (59), S16924 (48), buspirone (41), S15535 (22), clozapine (22), ziprasidone (21), pindolol (7), p-MPPI (0), WAY100,635 (0), spiperone (0). Despite differences in species and tissue source, the efficacy and potency (pEC₅₀) of agonists (with the exception of clozapine) correlated well with those determined previously at human 5-HT_1A receptors expressed in Chinese hamster ovary (CHO) cells. In contrast, clozapine was more potent at hippocampal membranes. The selective antagonists p-MPPI and WAY100,635 abolished stimulation of binding by (+)8-OH-DPAT, clozapine and S16924 (p-MPPI), indicating that these actions were mediated specifically by 5-HT_1A receptors. Clozapine and S16924 also attenuated 5-HT- and (+)8-OH-DPAT-stimulated [³⁵S]GTPS binding, consistent with partial agonist properties. In [³⁵S]GTPS autoradiographic studies, 5-HT-induced stimulation, mediated through 5-HT_1A receptors, was more potent in the septum (pEC₅₀~6.5) than in the dentate gyrus of the hippocampus (pEC₅₀~5) suggesting potential differences in coupling efficiency or G protein expression. Though clozapine (30 and 100 µM) did not enhance [³⁵S]GTPS labelling in any structure, S16924 (10 µM) modestly increased [³⁵S]GTPS labelling in the dentate gyrus. On the other hand, both these antipsychotic agents attenuated 5-HT (10 µM)-stimulated [³⁵S]GTPS binding in the dentate gyrus and septum. In conclusion, clozapine, S16924 and ziprasidone act as partial agonists for G protein activation at postsynaptic 5-HT_1A receptors in the hippocampus. These data support a role of postsynaptic 5-HT_1A receptors in the functional profiles of certain antipsychotic agents.     

Age-related behavioural,neurochemical and radioligand binding changes in the central 5-HT system of Sprague-Dawley rats

Mature (3–4 months) and aged (18–19 months) Sprague-Dawley (SD) rats were treated with 5-HT receptor agonists and drug-induced behaviours monitored. The 5-HT_2/1C agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), induced wet dog shakes and back muscle contractions which were significantly increased in aged, compared to mature, rats, suggesting an age-related enhancement of 5-HT₂ receptor function. In contrast, the selective 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) induced forepaw treading, flat body posture, hypothermia and hyperactivity which were not significantly different in aged compared to mature rats. Levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus and frontal cortex were measured using high performance liquid chromatography with electrochemical detection. There were no age-related changes in hippocampal 5-HT or 5-HIAA. However both 5-HT and 5-HIAA were increased in the frontal cortex of aged SD rats. 8-OH-DPAT reduced 5-HIAA in both regions examined in mature rats, an effect which was attenuated in the aged rats, suggesting an age-related reduction in presynaptic 5-HT_1A receptor function. DOI did not induce any changes in 5-HT or 5-HIAA in either of the regions examined. Radioligand binding studies with [³H] ketanserin showed there to be no significant age-related changes in cortical 5-HT₂ receptor density or affinity. In the samples taken from mature rats GTP shifted the competition curve to DOI and reduced the proportion of high affinity agonist binding sites; this effect was not observed in the aged samples, suggesting that there may be age-related changes in G-protein-mediated receptor-effector coupling mechanisms.     

Effects of fenfluramine on free-operant timing behaviour: evidence for involvement of 5-HT<Subscript>2A</Subscript> receptors

Rationale Temporal differentiation in the free-operant psychophysical procedure is sensitive to the 5-hydroxytryptamine (5-HT)_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and the 5-HT₂ receptor agonist 2,5-dimethoxy-4-iodo-amphetamine (DOI); both drugs shift the psychophysical curve leftwards, reducing the indifference point, T ₅₀. We have examined the effect of the 5-HT releasing agent fenfluramine on temporal differentiation.Objective We examined whether fenfluramines effect on temporal differentiation can be antagonised by the 5-HT_1A receptor antagonist N-[2-(4-[2-methoxy-phenyl]-1-piperazinyl)ethyl]-N-2-pyridinylcyclohexane-carboxamide (WAY-100635) and the 5-HT_2A receptor antagonist ketanserin, and compared the effects of fenfluramine, DOI and 8-OH-DPAT in intact rats and rats whose 5-HTergic pathways had been destroyed by 5,7-dihydroxytryptamine.Methods Rats were trained under the free-operant psychophysical procedure to press levers A and B in 50-s trials in which reinforcers were provided intermittently for responding on A in the first half, and B in the second half of the trial. Percent responding on B (%B) was recorded in successive 5-s epochs of the trials; logistic psychophysical curves were fitted to the data for derivation of timing indices (T ₅₀, time corresponding to %B=50%, and Weber fraction). Experiment 1 examined the effects of acute treatment with fenfluramine, and the interaction between fenfluramine and the 5-HT_1A and 5-HT_2A receptor antagonists WAY-100635 and ketanserin; experiment 2 compared the effects of fenfluramine, 8-OH-DPAT and DOI in intact rats and rats whose 5-HTergic pathways had been destroyed by intra-raphe injection of 5,7-dihydroxytryptamine. Concentrations of 5-HT and catecholamines in the brain were measured by high-performance liquid chromatography.Results Experiment 1: fenfluramine (2 mg/kg) reduced T ₅₀; this effect was attenuated by ketanserin (1.0 mg/kg) but not by WAY-100635 (100 g/kg). Experiment 2: 8-OH-DPAT (100 g/kg) and DOI (250 g/kg) reduced T ₅₀ in both groups; fenfluramine reduced T ₅₀ only in the sham-lesioned group. Levels of 5-HT were reduced by 80% in the lesioned group; catecholamine levels were not affected.Conclusions The results suggest that fenfluramine affects temporal differentiation via the release of endogenous 5-HT which acts mainly on postsynaptic 5-HT_2A receptors.     

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.     

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     

Simultaneous blockade of 5-HT<Subscript>1A/B</Subscript> receptors and 5-HT transporters results in acute increases in extracellular 5-HT in both rats and guinea pigs: in vivo characterization of the novel 5-HT<Subscript>1A/B</Subscript> receptor antagonist/5-HT transport inhibitor SB-649915-B

Rationale The delay in onset and treatment resistance of subpopulations of depressed patients to conventional serotonin reuptake inhibitors has lead to new drug development strategies to produce agents with improved antidepressant efficacy. Objectives We report the in vivo characterization of the novel 5-HT_1A/1B autoreceptor antagonist/5-HT transporter inhibitor (6-[(1-{2-[(2-methyl-5-quinolinyl)oxy]ethyl}-4-piperidinyl)methyl]-2H-1,4-benzoxazin-3(4H)-one), SB-649915-B. Materials and methods Ex vivo binding was used to ascertain 5-HT_1A receptor and serotonin transporter occupancy. 8-OH-DPAT-induced hyperlocomotion and SKF-99101-induced elevation of seizure threshold were used as markers of central blockade of 5-HT_1A and 5-HT_1B receptors, respectively. In vivo electrophysiology in the rat dorsal raphe and microdialysis in freely moving guinea pigs and rats were used to evaluate the functional outcome of SB-649915-B. Results SB-649915-B (1–10 mg/kg p.o.) produced a dose-related inhibition of 5-HT_1A receptor radioligand binding and inhibited ex vivo [³H]5-HT uptake in both guinea pig and rat cortex. SB-649915-B (0.1–10 mg/kg p.o.) reversed both 8-OH-DPAT-induced hyperlocomotor activity and SKF-99101-induced elevation of seizure threshold in the rat, demonstrating in vivo blockade of both 5-HT_1A and 5-HT_1B receptors, respectively. SB-649915-B (0.1–3 mg/kg i.v.) produced no change in raphe 5-HT neuronal cell firing per se but attenuated the inhibitory effect of 8-OH-DPAT. Acute administration of SB-649915-B resulted in increases (approximately two- to threefold) in extracellular 5-HT in the cortex of rats and the dentate gyrus and cortex of guinea pigs. Conclusions Based on these data, one may speculate that the 5-HT autoreceptor antagonist/5-HT transport inhibitor SB-649915-B will have therapeutic efficacy in the treatment of affective disorders with the potential for a faster onset of action compared to current selective serotonin reuptake inhibitors.     

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.     

Unaltered 5-HT- and desipramine-sensitive [3H]imipramine binding and [3H]5-HT uptake in rat brain after chronic imipramine and norzimeldine treatment

Several reports have shown heterogeneity of [³H]imipramine binding to brain membranes. Recently, a high affinity and 5-HT sensitive [³H]imipramine binding site of protein nature, that was suggested to be identical to the substrate recognition site for 5-HT uptake, was demonstrated. Since most studies on the regulation of the [³H]imipramine binding sites by antidepressants have used desipramine displaceable binding, which is heterogenous in nature and contains binding not related to 5-HT uptake sites, the present report studies the possible effects of chronic (3 weeks) administration of imipramine or norzimeldine (10 mg/kg intraperitoneally twice daily) on 5-HT sensitive [³H]imipramine binding sites. For comparison, desipramine sensitive binding was also studied, as well as the physiological correlate 5-HT uptake. There were no changes in either [³H]imipramine binding or 5-HT uptake after the antidepressant treatment.Supported by the Swedish Medical Research Council Offprint requests to: J. Marcusson at Dept. of Geriatric Medicine     

Selective remodeling of rabbit frontal cortex: relationship between 5-HT<Subscript>2A</Subscript> receptor density and associative learning

Rationale Associative learning during classical trace eyeblink conditioning has been shown to be regulated by serotonin 5-HT_2A receptors and to be critically dependent on the integrity of frontal cortex. Chronic administration of 5-HT_2A ligands has been shown to produce a selective up- or down-regulation of 5-HT_2A receptors in frontal cortex.Objectives We examined whether alterations in 5-HT_2A receptor density had a functional significance with respect to associative learning.Methods Animals received chronic injections of LSD, BOL or MDL11,939 and 1 day later began classical trace conditioning of the eyeblink response.Results The density of 5-HT_2A receptors in frontal cortex was significantly increased at 1–4 days after the cessation of chronic injections of the selective 5-HT_2A receptor ligand MDL11,939. Rabbits demonstrated an enhancement of associative learning when training began at 1 day after cessation of chronic MDL11,939 injections, but acquired at the same rate as controls when training began at 8 days after cessation of injections, a time when receptor density had returned to control levels. Animals that began training 1 day after chronic injections of BOL or LSD, drugs that produce decreases in 5-HT_2A receptor density, demonstrated normal rates of acquisition.Conclusions These results indicate that increases in the density of 5-HT_2A receptors in frontal cortex are associated with increases in the rate of associative learning, and further support an important role for this receptor in cortical circuitry that mediates learning. More generally, these results suggest an approach for functional remodeling of brain regions in the adult animal.