5-HT3 receptors which modulate [3H]5-HT release in the guinea pig hypothalamus are not autoreceptors

The 5-HT₃ agonist 2-methyl-5-HT had previously been shown to enhance the electrically evoked release of [³H]5-HT from preloaded slices of the guinea pig brain. In the present study, 2-methyl-5-HT (1 μM) was also found to increase the K⁺ evoked release of [³H]5-HT from preloaded slices of the guinea pig hypothalamus and this effect was blocked by the selective 5-HT₃ antagonist ondansetron. In the presence of tetrodotoxin, the enhancement of the K⁺-evoked release of [³H]5-HT by 2-methyl-5-HT in hypothalamus slices was blocked, thus suggesting that the 5-HT₃ receptors mediating this effect are not located directly on 5-HT terminals. In agreement with this, 2-methyl5-HT did not alter the K⁺-evoked release of [³H]5-HT in a synaptosomal preparation of the same brain structure, even at a concentration 10-fold greater than that used in the slices. Taken together, these data indicate that these facilitatory 5-HT₃ receptors are not located on 5-HT terminals in the guinea pig hypothalamus and therefore are not autoreceptors. © 1993 Wiley-Liss, Inc.     

Involvement of GABA systems in acetylcholine release induced by 5-HT3 receptor blockade in slices from rat entorhinal cortex

The aim of the present study was to examine the role of 5-HT₃ receptors in spontaneous and K⁺-evoked acetylcholine (ACh) release from rat entorhinal cortex and striatal slices. The 5-HT3 receptor antagonists ondansetron and granisetron (0.01–10 μM) produced a concentration-dependent increase in both spontaneous and K⁺-evoked [³H]ACh release in the two brain regions studied. The release of ACh was Ca²⁺-dependent and tetrodotoxin-sensitive. 5-HT₃ receptor agonists, such as 2-methyl-5-HT and 1-phenylbiguanide, at concentrations up to 1 μM, did not show any intrinsic effect on [³H]ACh release in both rat brain regions. However, 2-methyl-5-HT, 1 μM, fully blocked the ondansetron-induced enhancement in both basal and K⁺-evoked ACh release, suggesting that 5-HT₃ through 5-HT₃ receptor activation, tonically inhibits ACh release. The possible implication of interposed inhibitory systems in ACh release after 5-HT₃ receptor blockade was subsequently analyzed. While the effect of ondansetron was not modified by haloperidol or naloxone, the GABA_A receptor antagonist bicuculline produced a marked potentiation of ACh release in the entorhinal cortex but not in the striatum. The results suggest that in this cortical area 5-HT activates 5-HT₃ receptors located on GABAergic neurons which in turn inhibit cholinergic function.     

Pre- and post-synaptic effects of the 5-HT3 agonist 2-Methyl-5-HT on the 5-HT system in the rat brain

Microiontophoretic applications of 5-HT and of the 5-HT₃ agonist 2-methyl-5-HT produced a current-dependent suppression of firing activity of both hippocampal (CA₁ and CA₃) and cortical neurons in anesthetized rats. Concomitant microiontophoretic applications of the 5-HT₃ antagonists BRL 46470A and S-zacopride, as well as their intravenous injection, did not antagonize the inhibitory effect of 5-HT and 2-methyl-5-HT. In contrast, the 5-HT_1A antagonist BMY 7378, applied by microiontophoresis or administered intravenously, significantly reduced the inhibitory action of 5-HT and 2-methyl-5-HT. The firing activity of dorsal raphe 5-HT neurons was also reduced by 5-HT, 2-methyl-5-HT and the 5-HT_1A agonist 8-OH-DPAT applied by microiontophoresis. While BRL 46470A (0.1 and 1 mg/kg, i.v.) did not antagonize the inhibitory effect of the three 5-HT agonists on 5-HT neuronal firing activity, only that of 8-OH-DPAT was attenuated by the 5-HT_1A antagonist (+) WAY 100135. R-zacopride significantly reduced the duration of suppression of firing activity of CA₃ pyramidal neurons induced by the electrical stimulation of the ascending 5-HT pathway, and this reducing effect was prevented by the three 5-HT₃/5-HT₄ antagonists renzapride, S-zacopride and tropisetron, but not by BRL 46470A. Finally, in in vitro superfusion experiments, both BRL 46470A and S-zacopride antagonized the enhancing action of 2-methyld-HT on the electrically-evoked release of [³H]-5-HT in both rat frontal cortex and hippocampus slices. These findings suggest that, in vivo, the suppressant effect of 2-methyl-5-HT on the firing activity of dorsal hippocampus pyramidal, somatosensory cortical, and dorsal raphe 5-HT neurons is not mediated by 5-HT₃ receptors, but rather by 5-HT_1A receptors. The attenuating effect of R-zacopride on the effectiveness of the stimulation of the ascending 5-HT pathway is not mediated by 5-HT₃ receptors. In contrast, in vitro, the enhancing action of 2-methyl-5-HT on the electrically-evoked release of [³H]5-HT in both frontal cortex and hippocampus slices is mediated by 5-HT₃ receptors. © 1995 Wiley-Liss, Inc.     

Increased binding at 5-HT1A, 5-HT1B, and 5-HT2A receptors and 5-HT transporters in diet-induced obese rats

5-Hydroxytryptamine (5-HT, serotonin), synthesized in midbrain raphe nuclei and released in various hypothalamic sites, decreases food intake but the specific 5-HT receptor subtypes involved are controversial. Here, we have studied changes in the regional density of binding to 5-HT receptors and transporters and the levels of tryptophan hydroxylase, in rats with obesity induced by feeding a palatable high-energy diet for 7 weeks. We mapped binding at 5-HT receptor subtypes and transporters using quantitative autoradiography and determined tryptophan hydroxylase protein levels by Western blotting. In diet-induced obese (DiO) rats, specific binding to 5-HT_1A receptors ([³H]8-OH-DPAT) was significantly increased in the dorsal and median raphe by 90% (P<0.01) and 132% (P<0.05), respectively, compared with chow-fed controls. 5-HT_1B receptor binding sites ([¹²⁵I]cyanopindolol) were significantly increased in the hypothalamic arcuate nucleus (ARC) of DiO rats (58%; P<0.05), as were 5-HT_2A receptor binding sites ([³H]ketanserin) in both the ARC (44%; P<0.05) and lateral hypothalamic area (LHA) (121%; P<0.05). However, binding to 5-HT_2C receptors ([³H]mesulgergine) in DiO rats was not significantly different from that in controls in any hypothalamic region. Binding to 5-HT transporters ([³H]paroxetine) was significantly increased (P<0.05) in both dorsal and median raphe, paraventricular nuclei (PVN), ventromedial nuclei (VMH), anterior hypothalamic area (AHA) and LHA of DiO rats, by 47%–165%. Tryptophan hydroxylase protein levels in the raphe nuclei were not significantly different between controls and DiO rats. In conclusion, we have demonstrated regionally specific changes in binding to certain 5-HT receptor subtypes in obesity induced by voluntary overeating of a palatable diet. Overall, these changes are consistent with reduced 5-HT release and decreased activity of the 5-HT neurons. Reduction in the hypophagic action of 5-HT, possibly acting at 5-HT_1A, 5-HT_1B and 5-HT_2A receptors, may contribute to increased appetite in rats presented with highly palatable diet.     

5-HT2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D₁ receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K⁺-evoked [³H]ACh release while SCH 23390, a dopamine D₁ receptor antagonist, had no effect. [³H]ACh release was decreased by the dopamine D₂ receptor agonist LY 171555 (quinpirole) and slightly potentiated by the dopamine D₂ receptor antagonist haloperidol. The selective neurokinin NK₁ receptor agonist [Sar⁹, met(O₂)¹¹]SP also potentiated K⁺-evoked release of [³H]ACh. GR 82334, a NK₁ receptor antagonist, blocked not only the effect of [Sar⁹, met(O₂)¹¹]SP but also the release of ACh induced by the D₁ receptor agonist SKF 38393. Among the 5-HT agents studied, only the 5-HT_2A receptor antagonists ketanserin and ritanserin were able to reduce the ACh release induced by dopamine D₁ receptor stimulation. Mesulergine, a more selective 5-HT_2C antagonist, showed an intrinsic releasing effect but did not affect K⁺-evoked ACh release induced by SKF 38393. Methysergide and methiothepin, mixed 5-HT_1/2 antagonists, as well as ondansetron, a 5-HT₃ receptor antagonist, showed an intrinsic effect on ACh release, their effects being additive to that of SKF 38393. 5-HT₂ receptor agonists were ineffective. However, the 5-HT₂ agonist DOI was able to prevent the antagonism by ketanserin of the increased [³H]ACh efflux elicited by SKF 38393, suggesting a permissive role of 5-HT_2A receptors. None of the above indicated 5-HT agents was able to reduce the ACh release induced by the selective NK₁ agonist. The results suggest that 5-HT₂ receptors, probably of the 5-HT_2A subtype, modulate the release of ACh observed in slices from the rat striatum after stimulation of dopamine D₁ receptors. It seems that this serotonergic control is exerted on the interposed collaterals of substance P-containing neurons which promote ACh efflux through activation of NK₁ receptors located on cholinergic interneurons.     

The effect of ibogaine on κ-opioid- and 5-HT3-induced changes in stimulation-evoked dopamine release in vitro from striatum of C57BL/6BY mice

Ibogaine is an kndole alkaloid that has bean suggested to have potential efficacy for internrpdng dependermy on stimulant drugs. The κ-opiold and serotonkr 5-HT₃ systems may be involved in the action of Ibogaine, related to their modulation of dopaminergic transmission. The κ-opioid agonist U 62066 attenuated the in vitrostimulation-evoked efflux of tritium label from striatal tissue prelabeled with (₃H)dopamine. In mice protreated with Ibogaine·HCl (40 mg/kg IP given 2 h prior or 2 × 40 mg/kg and animals killed 18 h later), the Inhibitory effect of U 62066 on stimulation-evoked release of tritium was eliminated. The 5-HT₃ agonist phernylbiguanide had a biphasic effect on stimulation-evoked release of tritium; at 10⁻⁶ M phenylbiguanide, stimulation-evoked release was attenuated. At 10⁻⁵ M the basal outflow of tritium was increased. Ibogaine pretreatment had no effect on basal or stimulation-evokedrelease in the presence of 10⁻⁶ M phenylbiguanide, but increased the stimulation-evoked outflow of bftkan in the presence of 10⁻⁵ M phenylbiguanide. Cocaine (10⁻⁶ M), a dopanlne uptake blocker, increased the electrically-evoked release of dopamine; ibogaine pretreatment did not affect the enhanced electrically-induced release of (³H]dopamine by in vitro cocalne. The effects of ibogaine on the κ-opioid and 5-HT₃ receptors, located presynaptically on stristal dopamine terminals, modulating dopamine release may partly underlie its putative antiaddictive properties.     

Effects of cholecystokinin tetrapeptide (CCK4) and anxiolytic drugs on the electrically evoked [H]5-hydroxytryptamine outflow from rat cortical slices

The outflow of [³H]5-hydroxytryptamine ([³H]5-HT) from electrically stimulated rat cortical slices was measured to ascertain the modulatory role of endogenous cholecystokinin (CCK) on the amine outflow and to test the hypothesis that different anxiolytic compounds inhibit 5-HT secretion. The [³H]5-HT outflow evoked at 10 Hz was increased up to +30% by CCK₄ 300–1000 nM, the effect being prevented by the CCK_B receptor antagonist GV 150013, 3 nM. The limited sensitivity to CCK₄ seemed to depend on 5-HT auto-receptor feedback because pre-treatment with 100 nM methiothepin enhanced the [³H]5-HT outflow and lowered the CCK₄ threshold concentration from 300 to 30 nM. In addition, pre-treatment with 1 μM bacitracin to inhibit CCK metabolism increased [³H]5-HT efflux. This effect was concentration-dependently counteracted by GV150013 suggesting the presence of an endogenous CCK positive modulation. GV150013 30 nM, the 5-HT_1A partial agonist buspirone 300 nM and the GABA_A receptor modulator diazepam 10 nM, known to have anxiolytic properties, all significantly reduced the [³H] amine outflow from cortical slices by about 20%. This inhibition depended on their interaction with their respective receptors, which seemed to restrain the activity of functionally interconnected glutamatergic interneurones. In fact, APV (50 μM) and NBQX (10 μM) prevented the effect of the anxiolytic compounds. Thus, anxiolytic drugs with different receptor targets can reduce 5-HT outflow by dampening the glutamatergic signal, and in turn, the secretory process of the serotonergic nerve ending.     

Release of [3H]5-hydroxytryptamine from the intermediate area of rat thoracic spinal cord is modulated by presynaptic autoreceptors

Serotonin (5-HT) nerve terminals innervate sympathetic preganglionic neurons of the intermediolateral cell column (IML); however, neither the depolarization-induced release of 5-HT nor the presence of presynaptic modulatory autoreceptors have been directly studied in this system. We used in vitro superfusion of the microdissected intermediate area (including the intermediolateral cell column, intercalated nucleus, and central autonomic nucleus) of the rat thoracic spinal cord to measure basal and stimulated release of preloaded [³H]5-HT. Elevated K⁺ evoked a concentration- and Ca²⁺ dependent release of [³H]5-HT. Exogenous 5-HT and the 5-HT_1B agonist, CGS-12066B, both decreased the K⁺-stimulated release of [³H]5-HT. A 5-HT_1B antagonist (methiothepin) blocked the 5-HT- and the CGS-12066B-induced inhibition of K⁺-evoked release of [³H]5-HT. A 5-HT_1A antagonist (NAN-190) did not alter the inhibitory actions of exogenous 5-HT. Moreover, a 5-HT_1A agonist (8-OH-DPAT), a 5-HT_2A/2C agonist [(+/-)-DOI hydrochloride], and a 5-HT₃ agonist (2-methyl-5-HT) did not alter the K⁺ evoked release of [³H]5-HT. These data demonstrate that 5-HT is released from the intermediate area of the rat thoracic spinal cord. The 5-HT receptor subtype involved in the inhibition of the evoked release of [³H]5-HT is of the 5-HT_1B subtype. These findings may help clarify the complex role of 5-HT in spinal regulation of the sympathetic nervous system. © 1994 Wiley-Liss, Inc

1 This article is US Government work and, as such, is in the public domain in the United States of America.

     

The effect of ibogaine on κ-opioid- and 5-HT3-induced changes in stimulation-evoked dopamine release in vitro from striatum of C57BL/6BY mice

Ibogaine is an kndole alkaloid that has bean suggested to have potential efficacy for internrpdng dependermy on stimulant drugs. The κ-opiold and serotonkr 5-HT₃ systems may be involved in the action of Ibogaine, related to their modulation of dopaminergic transmission. The κ-opioid agonist U 62066 attenuated the in vitrostimulation-evoked efflux of tritium label from striatal tissue prelabeled with (₃H)dopamine. In mice protreated with Ibogaine·HCl (40 mg/kg IP given 2 h prior or 2 × 40 mg/kg and animals killed 18 h later), the Inhibitory effect of U 62066 on stimulation-evoked release of tritium was eliminated. The 5-HT₃ agonist phernylbiguanide had a biphasic effect on stimulation-evoked release of tritium; at 10⁻⁶ M phenylbiguanide, stimulation-evoked release was attenuated. At 10⁻⁵ M the basal outflow of tritium was increased. Ibogaine pretreatment had no effect on basal or stimulation-evokedrelease in the presence of 10⁻⁶ M phenylbiguanide, but increased the stimulation-evoked outflow of bftkan in the presence of 10⁻⁵ M phenylbiguanide. Cocaine (10⁻⁶ M), a dopanlne uptake blocker, increased the electrically-evoked release of dopamine; ibogaine pretreatment did not affect the enhanced electrically-induced release of (³H]dopamine by in vitro cocalne. The effects of ibogaine on the κ-opioid and 5-HT₃ receptors, located presynaptically on stristal dopamine terminals, modulating dopamine release may partly underlie its putative antiaddictive properties.     

Blockade of 5-HT(2) receptors in the periaqueductal grey matter (PAG) abolishes the anxiolytic-like effect of 5-HT(1A) receptor antagonism in the median raphe nucleus in mice

Several lines of evidence support the involvement of serotonergic (5-HT) neurons of the median raphe nucleus (MRN) in anxiety-like behaviour. In this context, it is known that blockade of 5-HT_1A somatodendritic autoreceptors in the midbrain raphe nuclei increases the firing rate of these neurons, disinhibiting 5-HT release in postsynaptic target areas such as amygdala, hippocampus and periaqueductal grey matter (PAG). However, while activation of 5-HT_1A or 5-HT₂ receptors in forebrain targets such as the amygdala or hippocampus enhances anxiety-like behaviours in rodents, stimulation of both receptor subtypes in the midbrain PAG markedly reduces anxiety-like behaviour. In view of these findings, the present study investigated whether the anti-anxiety effects induced by pharmacological disinhibition of 5-HT neurons in the MRN are attenuated by the blockade of 5-HT₂ receptors within the PAG. Mice received combined intra-PAG injection with ketanserin (10 nmol/0.1 μl), a 5-HT₂ receptor antagonist, followed by intra-MRN injection of WAY-100635 (5.6 nmol/0.1 μl), a highly selective 5-HT_1A receptor antagonist. They were then individually exposed to the elevated plus-maze (EPM), with the videotaped behavioural sessions subsequently scored for both conventional and ethological measures. The results confirmed that intra-MRN infusion of WAY100635 reduces behavioural indices of anxiety without significantly altering general activity measures, and further showed that this effect was completely blocked by intra-PAG pretreatment with an intrinsically-inactive dose of ketanserin. Together, these results suggest that 5HT₂ receptor populations located within the midbrain PAG play a significant role in the reduction of anxiety observed following disinhibition of 5-HT neurons in the MRN.     

Correspondence between 5-HT2 receptors and serotonergic axons in rat neocortex

The anatomic relationship between serotonergic (5-HT) axons and 5-HT₂ receptors in the rat forebrain was determined by a combined analysis of transmitter immunocytochemistry and receptor autoradiography. High densities of 5-HT₂ receptors, localized by the ligand N1-methyl-2-¹²⁵I-LSD (¹²⁵I-MIL), are found in neocortex and striatum; these regions also receive a dense serotonergic innervation. Regional variations in the density of 5-HT₂ receptors and 5-HT axons correspond closely in most, but not all, areas of the forebrain. In somatosensory cortex (SI), the laminar distribution of 5-HT₂ receptors closely matches that of 5-HT axons: in particular, a dense band of 5-HT₂ receptors in layer V_a of SI is in precise register with a dense plexus of fine 5-HT axons. We have also observed a close spatial relationship between 5-HT₂ receptors and fine axons in other areas of the forebrain, suggesting that 5-HT₂ receptors may be selectively linked to a particular type of 5-HT axon terminal. Since fine axons of this type have been reported to arise from the dorsal raphe nucleus, it appears likely that 5-HT₂ receptors may mediate the effects of dorsal but not median raphe projections.     

Pharmacological characterization of 5-hydroxytryptamine receptor types in the guinea-pig proximal colon

It was investigated which 5-hydroxytryptamine (5-HT) receptors mediate the responses to 5-HT in the longitudinal muscle layer of the guinea-pig proximal colon, using selective 5-HT receptor antagonists and the 5-HT analogues α-methyl-5-HT (2-Me-5-HT), 2-methyl-5-HT (2-Me-5-HT), and 5-methoxytryptamine (5-MeOT). 5-HT as well as its analogues induced concentration-related contractions, at low concentrations preceded by relaxations. The 5-HT concentration-contractile response curve was biphasic whilst the curves to α-Me-5-HT, 2-Me-5-HT, and 5-MeOT were monophasic. Tetrodotoxin (TTX) abolished the relaxations, and it inhibited the contractions to all agonists. In the presence of TTX, blockade of either 5-HT₂ (ketanserin) or 5-HT₃ receptors (ondansetron, tropisetron) reduced the contractions to 5-HT, whereas blockade of both 5-HT receptor types at the same time abolished them. In the absence of TTX, the contractions to 5-HT were inhibited by antagonists at 5-HT₂ (ketanserin), 5-HT₃ (granisetron, nanomolar concentration of tropisetron) and also 5-HT₄ receptors (micromolar concentration of tropisetron). Contractions to α-Me-5-HT did not seem to be sensitive to 5-HT₂ receptor blockage with ketanserin, but in the presence of TTX the contractions were abolished by the 5-HT₂ receptor antagonist. The 5-HT₃ receptor antagonist granisetron abolished contractions to 2-Me-5-HT. In the presence of TTX, the 5-HT₂ receptor antagonist ketanserin abolished contractions to 5-MeOT, and in the absence of TTX the contractions to 5-MeOT were highly sensitive blockade of 5-HT₄ receptors with tropisetron. Blockage of either 5-HT₁ (methiothepin), 5-HT₂ (ketanserin), 5-HT₃ (ondansetron, granisetron, tropisetron) or 5-HT₄ (tropisetron) receptors did not abolish the relaxations to 5-HT or 5-MeOT. In conclusion, 5-HT induces contractions of the longitudinal muscle of the guinea-pig proximal colon, through the stimulation of 5-HT₂ receptors on the smooth muscle cells and 5-HT₃ receptors and putative neuronal 5-HT₄ receptors. 5-HT evokes relaxations via an unknown neuronal receptor.     

Effects of adrenalectomy and type I or type II glucocorticoid receptor activation on 5-HT1A and 5-HT2 receptor binding and 5-HT transporter mRNA expression in rat brain

We evaluated the effects of adrenalectomy (ADX) and replacement with glucocorticoid receptor agonists on serotonin (5-HT) 5-HT_1A and 5-HT₂ receptor binding in rat brain. 5-HT_1A receptor binding was increased in the CA2–CA4 and the dentate gyrus of the hippocampus 1 week after ADX. This effect was prevented by the systemic administration of aldosterone (10 μg/μl/h) but not by RU28362 (10 μg/μl/h). No significant effect was observed on 5-HT₂ receptor binding in rat cortex. The expression of 5-HT transporter mRNA was unchanged in the raphe nucleus as measured by in situ hybridization.     

Stimulation of the rat dorsal raphe in vivo releases labeled serotonin from the parietal cortex

In vivo release of labeled serotonin ([³H]5-HT) from the parietal cortex was investigated by cortical cup technique and electrical stimulation of midbrain raphe in rats anesthetized with pentobarbital sodium. The spontaneous efflux of tritium from the parietal cortex preloaded with [³H]5-HT followed a multiphasic exponential course. After 120 min, the rate of efflux appeared to fit the single exponential function (slow phase). Imipramine (10⁻⁶−10⁻³ M) produced a dose-dependent increase in the spontaneous release. When pargyline in concentrations ranging from 10⁻⁴ to 10⁻³ M were added to the medium in the cup, the unchanged [³H]5-HT significantly increased in a dose-dependent manner and the slow declining coefficient of tritium efflux significantly decreased in the presence of 10⁻⁴ pargyline. Stimulation of the rostral two-thirds of the dorsal raphe and the lateral 5-HT bundle originating from the dorsal raphe significantly increased the release of [³H]5-HT and its metabolites while stimulation of the caudal one-third of the dorsal raphe did not produce a significant increase in the release of [³H]5-HT and its metabolites. Stimulation of the median raphe produced no or only a slight increase in the release of [³H]5-HT and its metabolites. These findings are a direct demonstration of the in vivo release of [³H]5-HT from the parietal cortex with stimulation of the dorsal raphe, particularly the rostral two-thirds of the nucleus and provide the neurochemical evidence for the dorsal raphe-cortical 5-HT pathway via the lateral 5-HT bundle.     

5-HT1A and 5-HT2 receptors differentially regulate the excitability of 5-HT-containing neurones of the guinea pig dorsal raphe nucleus in vitro

We have used intracellular recording techniques to examine the effects of 5-hydroxytryptamine (5-HT, serotonin) on 5-HT-containing neurones of the guinea pig dorsal raphe nucleus in vitro. Bath-applied 5-HT (30–300 μM) had two opposing effects on the membrane excitability of these cells, reflecting the activation of distinct 5-HT receptor subtypes. As demonstrated previously in the rat, 5-HT evoked a hyperpolarization and inhibition of 5-HT neurones, which appeared to involve the activation of an inwardly rectifying K⁺ conductance. This hyperpolarizing response was blocked by the 5-HT_1A receptor-selective antagonist WAY-100635 (30–100 nM). In the presence of WAY-100635, 5-HT induced a previously unreported depolarizing, excitatory response of these cells, which was often associated with an increase in the apparent input resistance of the neurone, likely due to the suppression of a K⁺ conductance. Like the hyperpolarizing response to 5-HT, this depolarization could be recorded in the presence of the Na⁺ channel blocker tetrodotoxin. In addition, the response was not significantly attenuated by the α₁-adrenoceptor antagonist prazosin (500 nM), indicating that it is not due to the release of noradrenaline, or to the direct activation of α₁-adrenoceptors by 5-HT. The 5-HT₃ receptor antagonist granisetron (1 μM) and the 5-HT₄ receptor antagonist SB 204070 (100 nM) failed to reduce the depolarizing response to 5-HT; however, ketanserin (100 nM), mesulergine (100 nM) and lysergic acid diethylamide (1 μM) significantly reduced or abolished the depolarization, indicating that this effect of 5-HT is mediated by 5-HT₂ receptors.     

Functional evidence for the rapid desensitization of 5-HT3 receptors on vagal afferents mediating the Bezold–Jarisch reflex

The aim of this study was to determine whether 5-hydroxytryptamine (5-HT)₃ receptors on cardiopulmonary afferents mediating the Bezold–Jarisch reflex (BJR) desensitize upon repeated exposure to selective agonists. BJR-mediated falls in heart rate, diastolic arterial blood pressure and cardiac output elicited by the 5-HT₃-receptor agonists, phenylbiguanide (100 μg/kg, i.v.) or 2-methyl-5-HT (100 μg/kg, i.v.), progressively diminished upon repeated injection in conscious rats. The BJR responses elicited by 5-HT (40 μg/kg, i.v.) were markedly reduced in rats which had received the above injections of phenylbiguanide or 2-methyl-5-HT whereas the BJR responses elicited by -S-nitrosocysteine (10 μmol/kg, i.v.) were similar before and after the injections of the 5-HT₃ receptor agonists. These findings suggest that tachyphylaxis to 5-HT₃ receptor agonists may be due to the desensitization of 5-HT₃ receptors on cardiopulmonary afferents rather than the impairment of the central or peripheral processing of the BJR.     

Evidence for 5-HT4 receptor involvement in the enhancement of acetylcholine release by p-chloroamphetamine in rat frontal cortex

The roles of endogenous serotonin (5-HT) and 5-HT receptor subtypes in regulation of acetylcholine (ACh) release in frontal cortex of conscious rats were examined using a microdialysis technique. Systemic administration (1 and 3 mg/kg, i.p.) of the 5-HT-releasing agent p-chloroamphetamine (PCA) elevated ACh output in a dose-dependent manner. Depletion of endogenous 5-HT by p-chlorophenylalanine significantly attenuated the facilitatory effect of PCA on ACh release. The PCA (3 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT₄ receptor antagonists RS23597 (50 μM) and GR113803 (1 μM), while the 5-HT_1A antagonist WAY-100135 (10 mg/kg, i.p.; 100 μM), 5-HT_1A/1B/β-adrenoceptor antagonists (−)-pindolol (8 mg/kg, i.p.) and (−)-propranolol (150 μM), 5-HT_2A/2C antagonist ritanserin (1 mg/kg, i.p.; 10 μM) and 5-HT₃ antagonist ondansetron (1 mg/kg, i.p.; 10 μM) failed to significantly modify the effect of PCA. These results suggest that PCA-induced enhancement of 5-HT transmission facilitates ACh release from rat frontal cortex at least in part through 5-HT₄ receptors.     

Characterization and quantitative autoradiography of [H]tryptamine binding sites in rat brain

[³H]tryptamine binds with high affinity (K_d = 9.1nM, B_max= 54fmol/mg wet wt.) to tissue sections of rat brain. The binding occurs rapidly and is reversible. Low concentrations of the β-carbolines harmaline (IC₅₀ = 25nM) and tetrahydronorharman (tetrahydro-β-carboline), IC₅₀ = 50nM) inhibit [³H]tryptamine binding. Serotonin (5-HT, IC₅₀ = 2600nM) as well as the 5-HT receptor antagonists methysergide and metergoline displace [³H]tryptamine at much higher concentrations from brain slices. The distribution of [³H]tryptamine binding sites in section of rat brain has been analyzed by quantitative autoradiography. The highest density of binding sites is found in the nucleus (n.) interpreduncularis, a slightly lower one in the locus coeruleus. Moderately labelled are the n. accumbens septi, n. septi lateralis, n. medalis habenulae, n. tractus olfactorii lateralis, the central region of the amydgala, n. caudatsu/putamen, n. reuniens and the hippocampal formation. A low density of binding sites is detected in the cerebral cortex and the subiculum. Even less binding sites are found in the n. dorsalis raphe and the substantia nigra. The pattern of distribution of [³H]tryptamine binding sites differs from that of [³H]5-HT(5-HT₁), [³H]ketanserin (5-HT₂) as well as [³H]imipramine binding sites. These data suggest unique tryptamine binding sites.     

Influence of serotoninergic drugs on in vivo dopamine extracellular output in rat striatum

In vivo microdialysis was used to investigate the mechanism behind the increase in extracellular dopamine (DA) induced by increase in extracellular serotonin (5-HT) level and 5-HT₁ and 5-HT₂ receptor activation. The following serotoninergic drugs were perfused in the absence or presence of nomifensine (5 μM)or tetrodotoxin (TTX; 2 μM): clomipramine (10, 500 and 1,000 μM), a selective 5-HT reuptake inhibitor; 8-OH-DPAT (50 and 500 μM), a 5-HT_1A receptor agonist; and α-methyl-5-HT (1, 5 and 50 μM), a 5-HT₂ receptor agonist. All the serotoninergic drugs studied increased DA extracellular output in a dose-dependent manner. The presence of nomifensine attenuated the effect of perfusion of clomipramine (500 μM) and completely abolished the effect of perfusion of 8-OH-DPAT (500 μM) and α-methyl-5-HT (5 μM) on DA extracellular output. Clomipramine (100–1,000 μM) perfusion produced a dose dependent increase in DOPAC extracellular output, which was stronger when clomipramine (500 μM) was co-perfused with nomifensine. 8-OH-DPAT and α-methyl-5-HT perfusion decreased DOPAC overflow. Addition of TTX to the perfusion fluid one hour before serotoninergic drugs perfusion, did not completely abolish the effect on dopamine extracellular output produced by the serotoninergic drugs. These data seem to indicate that increase in extracellular 5-HT level and 5-HT₁ and 5-HT₂ receptor activation increase in vivo DA extracellular output in the striatum mainly by a non-exocytotic mechanism involving DA uptake sites and, secondarily, by activation of 5-HT receptors. J. Neurosci. Res. 52:591–598, 1998. © 1998 Wiley-Liss, Inc.     

N-Methyl-d-aspartate receptors regulate 5-HT release in the raphe nuclei and frontal cortex of freely moving rats: differential role of 5-HT1A autoreceptors

The effects of infusing N-methyl-

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-aspartate (NMDA) into the raphe nuclei on release of 5-HT in this brain region and also the frontal cortex of the same animal were studied using in vivo microdialysis in freely moving rats. Infusion of 25 μM NMDA into the raphe led to a substantial decrease in dialysate 5-HT in this region and a prolonged increase in terminal 5-HT release in the frontal cortex. These effects were blocked by the specific NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (D-AP5; 100 μM). When 25 μM NMDA was co-infused into the raphe with the selective 5-HT_1A receptor antagonist (N-{2-{4-(2-methoxyphenyl)-1-piperazinyl}ethyl-N-(2-pyridinyl)cyclohexanecarboxamide) (WAY-100635; 1.0 μM) the effect of NMDA infusion was unaltered. WAY-100635 infused alone into the raphe did not alter local 5-HT or extracellular 5-HT in the cortex. Infusion of 100 μM NMDA into the raphe was followed by an increase in local dialysate 5-HT and a decrease in 5-HT release in the cortex. These changes were reversed by D-AP5. Following infusion of 100 μM NMDA with 1.0 μM WAY-100635 into the raphe local 5-HT release was still increased, however, the decrease in 5-HT observed in the frontal cortex was abolished. These data suggest that the degree of NMDA receptor activation leads to dramatically different outcomes with regard to serotonergic transmission to the frontal cortex. Furthermore, there appears to be a differential role of the 5-HT_1A autoreceptor in regulating these effects. These data are discussed in relation to other studies on the regulation of serotonergic transmission in ascending pathways.