Characterisation of the selective 5-HT1B receptor antagonist SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride): in vivo neurochemical and behavioural evidence of anxiolytic/antidepressant activity

The 5-HT_1B receptor has attracted significant interest as a potential target for the development of therapeutics for the treatment of affective disorders such as anxiety and depression. Here we present the in vivo characterisation of a novel, selective and orally bioavailable 5-HT_1B receptor antagonist, SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride). SB-616234-A reversed the 5-HT_1/7 receptor agonist, SKF-99101H-induced hypothermia in guinea pigs in a dose related manner with an ED₅₀ of 2.4 mg/kg p.o. Using in vivo microdialysis in freely moving guinea pigs, SB-616234-A (3–30 mg/kg p.o.) caused a dose-related increase in extracellular 5-HT in the dentate gyrus. Evaluation of antidepressant- and anxiolytic-like effects of this 5-HT_1B receptor antagonist was performed in a variety of models and species. SB-616234-A produced a decrease in immobility time in the mouse forced swim test; an effect suggestive of antidepressant activity. Furthermore, SB-616234-A produced dose-related anxiolytic effects in both rat and guinea pig maternal separation-induced vocalisation models with an ED₅₀ of 1.0 and 3.3 mg/kg i.p., respectively (vs fluoxetine treatment ED₅₀ = 2.2 mg/kg i.p. in both species). Also a significant reduction in posturing behaviours was observed in the human threat test in marmosets; an effect indicative of anxiolytic activity. In summary, SB-616234-A is a novel, potent and orally bioavailable 5-HT_1B receptor antagonist which exhibits a neurochemical and behavioural profile that is consistent with both anxiolytic- and antidepressant-like activity in a variety of species. Taken together these data suggest that SB-616234-A may have therapeutic efficacy in the treatment of affective disorders.     

Biochemical evidence for the 5-HT agonist properties of PAT (8-hydroxy-2-(di-n-propylamino)tetralin) in the rat brain

In vitro investigations revealed that PAT (8-hydroxy-2-(n-dipropylamino)tetralin) interacted with postsynaptic 5-HT receptors in the rat brain: the drug stimulated 5-HT-sensitive adenylate cyclase in homogenates of colliculi from new-born rats (K_Aapp 8.6 μM) and inhibited the specific binding of [³H]5-HT to 5-HT₁ sites. The PAT-induced inhibition of [³H]5-HT binding showed marked regional differences compatible with a preferential interaction of PAT (IC₅₀ 2 nM) with the 5-HT_1A subclass. As previously seen with 5-HT agonists, the efficacy of PAT for displacing [³H]5-HT bound to hippocampal membranes was markedly increased by Mn²⁺ (1 nM) and reduced by GTP (0.1 nM). PAT also affected presynaptic 5-HT metabolism since it inhibited competitively (K_i 1.4 μM) [³H]5-HT uptake into cortical synaptosomes and reduced (in the presence of the 5-HT uptake inhibitor fluoxetine) the K⁺-evoked release of [³H]5-HT previously taken up or newly synthesized from [³H]tryptophan in cortical or striatal slices. This latter effect was prevented by 5-HT antagonists (methiothepin, metergoline) suggesting that it was mediated by the stimulation of presynaptic 5-HT autoreceptors by PAT. Like 5-HT, PAT counteracted the stimulatory effect of K⁺-induced depolarization on the synthesis of [³H]5-HT from [³H]tryptophan in cortical slices. It is concluded that PAT is a potent 5-HT agonist acting on both post- and presynaptic 5-HT receptors in the rat brain.     

SB-616234-A (1-[6-(cis-3,5-dimethylpiperazin-1-yl)-2,3-dihydro-5-methoxyindol-1-yl]-1-[2'methyl-4'-(5-methyl-1,2,3-oxadiazol-3-yl)biphenyl-4-yl]methanone hydrochloride): a novel, potent and selective 5-HT1B receptor antagonist

SB-616234-A possesses high affinity for human 5-HT_1B receptors stably expressed in Chinese hamster ovary (CHO) cells (pK_i 8.3 ± 0.2), and is over 100-fold selective for a range of molecular targets except h5-HT_1D receptors (pK_i 6.6 ± 0.1). Similarly, affinity (pK_i) for rat and guinea pig striatal 5-HT_1B receptors is 9.2 ± 0.1. In [³⁵S]-GTPγS binding studies in the human recombinant cell line, SB-616234-A acted as a high affinity antagonist with a pA₂ value of 8.6 ± 0.2 whilst providing no evidence of agonist activity in this system. In [³⁵S]-GTPγS binding studies in rat striatal membranes, SB-616234-A acted as a high affinity antagonist with an apparent pK_B of 8.4 ± 0.5, again whilst providing no evidence of agonist activity in this system. SB-616234-A (1 μM) potentiated electrically stimulated [³H]-5-HT release from guinea pig and rat cortical slices (S₂/S₁ ratios of 1.8 and 1.6, respectively). SB-616234-A (0.3–30 mg kg⁻¹ p.o.) caused a dose-dependent inhibition of ex vivo [³H]-GR125743 binding to rat striatal 5-HT_1B receptors with an ED₅₀ of 2.83 ± 0.39 mg kg⁻¹ p.o. Taken together these data suggest that SB-616234-A is a potent and selective 5-HT_1B autoreceptor antagonist that occupies central 5-HT_1B receptors in vivo following oral administration.     

5-HT2/5-HT1C receptor-mediated facilitatory action on unit activity of ventral horn cells in rat spinal cord slices.

5-Methoxy-N,N-dimethyltryptamine (5-McODMT) and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) facilitate motoneuron excitability through 5-HT_1C/5-HT₂ receptors in rats. Using spinal cord slices prepared from adult rals, we recorded unitary cell discharges, evoked by local stimulation of the adjacent site, extracellularly in the motor nuclei of the ventral horn. 5-MeODMT, DOI, 5-hydroxytryptamme (5-HT), 8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) and tandospirone facilitated the probability of firing in the motor nuclei, with 5-MeODMT and DOI being the most potent. The effect of 5-MeODMT was significantly suppressed by ketanserin (a 5-HT₂ receptor-selective antagonist), spinerone (a 5-HT_1A/5-HT₂ receptor antagonist) and cyproheptadine (a 5-HT_1A/5-HT₂ receptor antagonist), but not by 3-tropanyl-3,5-dichlorobenzoate (MDL 72222, a 5-HT₃ receptor-selective antagonist) or pindolol (a 5-HT_1A/5-HT_1B receptor antagonist). This suggests that 5-HT₂ and/or 5-HT_1C receptors are involved in the facilitatory effects of 5-HT receptor agonists on the synaptic activity of ventral horn cells.     

Characterization of 5-hydroxytryptamine-induced depolarizations in rat isolated vagus nerve

An additional component of the depolarization induced by 5-hydroxytryptamine (5-HT) in the rat isolated vagus nerve has recently been attributed to activation of 5-HT₄ receptors. To confirm and extend this finding, extracellular recordings of D.C. potentials were made using the ‘grease-gap’ technique during continuous superfusion of the isolated nerve. Beginning at 1 nM, 5-HT induced small depolarizations that displayed a slow onset. At concentrations 1 μM, large depolarizations with rapid onset were elicited. In the presence of the 5-HT₃ receptor antagonists, granisetron or ondansetron, 5-HT responses were diminished and exhibited an increased latency to peak. These small, slow depolarizations were not reduced by 5-HT₁ or 5-HT₂ receptor antagonists, but were potently inhibited by the 5-HT₄ receptor antagonist GR 113808 (pA₂ = 9.3), and mimicked by 5-methoxytryptamine (pEC₅₀ = 5.3). 5-HT₄-mediated responses were larger at 37°C than at 31°C, but also showed marked diminution with repeated 5-HT applications at concentrations greater than 1 μM. Conversely, 5-HT₃ receptor responses were potentiated at lower temperatures (31°C). Consistent with the reported positive coupling of 5-HT₄ receptors to adenylyl cyclase, forskolin and 8-Br-cAMP produced slowly developing depolarizations which were qualitatively similar to 5-HT₄ receptor activation. Pre-depolarization of nerves with 10 μM forskolin or 300 μM 8-Br-cAMP diminished the effect of 5-HT₄ receptors. This study has confirmed the presence of 5-HT₄ receptors on the vagus nerve of the rat and defined some conditions that optimize their pharmacological isolation. The rat isolated vagus nerve constitutes a simple and robust preparation for studying 5-HT₄ receptors in the peripheral nervous system.     

Potent antagonism of 5-HT(3) and 5-HT(6) receptors by olanzapine.

The interaction of the psychotropic agent olanzapine with serotonin 5-HT₃ and 5-HT₆ receptors was investigated. Olanzapine did not contract the isolated guinea pig ileum, but blocked contractions induced by the 5-HT₃ receptor agonist 2-methyl serotonin (2-CH₃ 5-HT) with a pK_B value of 6.38±0.03, close to the affinity of the 5-HT₃ receptor antagonist ondansetron. The atypical antipsychotic risperidone (1 μM) did not significantly inhibit 2-CH₃ 5-HT-induced contractions. Olanzapine had high affinity (pK_i=8.30±0.06) for human 5-HT₆ receptors in radioligand binding studies. Olanzapine did not stimulate [³⁵S]guanosine-5′-O-(3-thio)triphosphate ([³⁵S]GTPγS) binding to the G protein G_s in cells containing human 5-HT₆ receptors, but inhibited 5-HT-stimulated [³⁵S]GTPγS binding (pK_B=7.38±0.16). Among other antipsychotics investigated, clozapine antagonized 5-HT₆ receptors with a pK_B=7.42±0.15, ziprasidone was three-fold less potent, and risperidone, quetiapine and haloperidol were weak antagonists. Thus, olanzapine was not an agonist, but was a potent antagonist at 5-HT₆ receptors and had marked antagonism at 5-HT₃ receptors.     

Selective labelling of 5-HT7 receptor recognition sites in rat brain using [H]5-carboxamidotryptamine

The aim of the present study was to establish a radioligand binding assay to selectively label the native 5-HT₇ receptor expressed in rat brain. In rat whole brain (minus cerebellum and striatum) homogenate, (±)-pindolol (10 μM)-insensitive [³H]5-CT ([³H]5-carboxamidotryptamine; 0.5 nM) specific binding (defined by 5-HT, 10 μM) displayed a pharmacological profile similar to the recombinant 5-HT₇ receptor, although the Hill coefficients for competition curves generated by methiothepin, ritanserin, sumatriptan, clozapine and pimozide were significantly less than unity. In homogenates of rat hypothalamus, (±)-pindolol (10 μM)-insensitive [³H]5-CT recognition sites also resembled, pharmacologically, the 5-HT₇ receptor, although pimozide still generated Hill coefficients significantly less than unity. Subsequent studies were performed in the additional presence of WAY100635 (100 nM) to prevent [³H]5-CT binding to residual, possibly, 5-HT_1A sites. Competition for this [³H]5-CT binding indicated the labelling in whole rat brain homogenate of a homogenous population of sites with the pharmacological profile of the 5-HT₇ receptor. Saturation studies also indicated that (±)-pindolol (10 μM)/WAY 100635 (100 nM)-insensitive [³H]5-CT binding to homogenates of whole rat brain was saturable and to an apparently homogenous population of sites which were labelled with nanomolar affinity (B_max=33.2±0.7 fmol mg⁻¹ protein, pK_d=8.78±0.05, mean±S.E.M., n=3). The development of this 5-HT₇ receptor binding assay will aid investigation of the rat native 5-HT₇ receptor.     

Pharmacological analysis of the variation in behavioural responses to tryptamine in five strains of mice

The effects of tryptamine on behaviour were studied in five strains of mice. There were significant strain differences in the intensity of the 5-HT syndrome (head weaving and hindlimb abduction) and head twitch responses. The intensity of the 5-HT syndrome correlated significantly with the brain tryptamine content, although the occurrence of head twitch was unrelated. The 5-HT₂ receptor antagonist, ketanserin, antagonized the head twitch responses elicited by tryptamine without affecting the head weaving or hindlimb abduction. Metergoline, an antagonist of both the 5-HT₁ and 5-HT₂ receptors, strongly inhibited both the 5-HT syndrome and the head twitch responses in all five strains. [³H]5-HT and [³H]ketanserin binding in the frontal cortex of the five strains of mice was also studied. The number of [³H]5-HT binding sites did not differ among the five strains of mice although there was a noticeable difference in the number of [³H]ketanserin binding sites. These results suggest that the 5-HT syndrome and the head twitch responses induced by tryptamine are linked separately with the 5-HT₁ and 5-HT₂ receptors, respectively. The strain differences in the tryptamine-induced 5-HT syndrome can be explained by the different levels of brain tryptamine. The differences in the head twitch responses in the mice are probably derived from the differences in the 5-HT₂ receptor binding sites in the brain and the content of brain tryptamine.     

Phencyclidine-induced cognitive deficits in mice are improved by subsequent subchronic administration of the antipsychotic drug perospirone: Role of serotonin 5-HT1A receptors

Accumulating evidence suggests that the serotonin 5-HT_1A receptor may play a role in the pathophysiology of schizophrenia. The present study was undertaken to examine the effects of perospirone, an atypical antipsychotic drug with 5-HT_1A receptor agonism, on cognitive deficits in mice after repeated administration of the NMDA receptor antagonist phencyclidine (PCP). Subsequent subchronic (14 days) administration of perospirone (1.0, 3.0, or 10 mg/kg) significantly attenuated PCP (10 mg/kg)-induced cognitive deficits in mice, in a dose-dependent manner. The effects of perospirone (10 mg/kg) were significantly antagonized by co-administration of the selective 5-HT_1A receptor antagonist WAY100635 (1.0 mg/kg). Furthermore, hypothermia by the 5-HT_1A receptor agonist 8-OH DPAT (0.25 mg/kg) was significantly attenuated in mice treated with PCP. Moreover, a receptor binding assay using [³H]WAY100635 revealed that levels of 5-HT_1A receptors in the hippocampus, but not in the frontal cortex, of PCP-treated mice were significantly lower than those of saline-treated mice. These findings suggest that repeated PCP administration alters 5-HT_1A receptor function in the mouse brain, and that subsequent subchronic administration of perospirone ameliorates PCP-induced cognitive deficits via 5-HT_1A receptors. Therefore, perospirone could be a potential therapy for the cognitive deficits observed in schizophrenic patients.     

5-Fluorotryptamine is a partial agonist at 5-HT3 receptors, and reveals that size and electronegativity at the 5 position of tryptamine are critical for efficient receptor function

Antagonists, but not agonists, of the 5-HT₃ receptor are useful therapeutic agents, and it is possible that partial agonists may also be potentially useful in the clinic. Here we show that 5-fluorotryptamine (5-FT) is a partial agonist at both 5-HT_3A and 5-HT_3AB receptors with an R_max (I_max / I_max5-HT) of 0.64 and 0.45 respectively. It is about 10 fold less potent than 5-HT: EC₅₀ = 16 and 27 μM, and K_i for displacement of [³H]granisetron binding = 0.8 and 1.8 μM for 5-HT_3A and 5-HT_3AB receptors respectively. We have also explored the potencies and efficacies of tryptamine and a range of 5-substituted tryptamine derivatives. At 5-HT_3A receptors tryptamine is a weak (R_max = 0.15), low affinity (EC₅₀ = 113 μM; K_i = 4.8 μM) partial agonist, while 5-chlorotryptamine has a similar affinity to 5-FT (EC_50 = 8.1 μM; K_i = 2.7 μM) but is a very weak partial agonist (R_max = 0. 0037). These, and data from 5-methyltryptamine and 5-methoxytryptamine, reveal the importance of size and electronegativity at this location for efficient channel opening.     

Involvement of 5-hydroxytryptamine1A receptors in Delta9-tetrahydrocannabinol-induced catalepsy-like immobilization in mice

The present study investigated the involvement of 5-hydroxytryptamine_1A (5-HT_1A) receptors in Δ⁹-tetrahydrocannabinol (THC)-induced catalepsy-like immobilization in mice. THC (10 mg/kg, i.p.) induced catalepsy-like immobilization but had no effect on motor coordination in the rota-rod test. The selective cannabinoid CB₁ receptor antagonist rimonabant (3 mg/kg, i.p.) completely antagonized THC-induced catalepsy-like immobilization. The 5-HT_1A/5-HT₇ receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT; 0.3 and 1 mg/kg, i.p.) and 5-HT_1A receptor partial agonist buspirone (0.06 and 0.1 mg/kg, i.p.) inhibited this THC-induced catalepsy-like immobilization. Moreover, the selective 5-HT_1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohezane carboxamide dihydrochloride (WAY100635; 0.3 or 1 mg/kg, i.p.) reversed the inhibition of THC-induced catalepsy-like immobilization by 8-OH-DPAT (1 mg/kg) or buspirone (0.06 mg/kg). In contrast, the selective 5-HT₇ receptor antagonist (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulfonyl]phenol hydrochloride (SB269970) had no effect on this inhibitory effect of 8-OH-DPAT. On the other hand, WAY100635 (0.3 and 1 mg/kg, i.p.) enhanced the catalepsy-like immobilization induced by THC (6 mg/kg, i.p.). These findings suggest that the 5-HT_1A receptors are involved in THC-induced catalepsy-like immobilization.     

Effect of acute administration of various 5-HT receptor agonists on focal hippocampal seizures in freely moving rats

In this study, we assessed the effects of the acute administration of various 5-HT receptor agonists on hippocampal partial seizures generated by low-frequency electrical stimulation in male Wistar rats. The seizure threshold and severity were determined by measuring the pulse number threshold and primary and secondary afterdischarges and the latency of secondary discharge was also determined. The administration (0.1–1 mg/kg, i.p.) of either the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-aminopropyl)tetralin (8-OH-DPAT), or the selective 5-HT₃ receptor agonist, 4-amino-(6-chloro-2-pyridyl)-1-piperidine (SR 57227A, 0.3–3 mg/kg, i.p.), did not alter any of the seizure parameters compared to those in vehicle-treated animals. Similarly, the administration of 0.3 and 1 mg/kg, i.p., of the 5-HT_2A,C receptor agonist, (±)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), did not alter any of the seizure parameters, whereas 3 mg/kg significantly decreased the latency of the secondary afterdischarge compared to that in vehicle-treated animals. The selective serotonin reuptake inhibitor, (±)-fluoxetine (2 mg/kg, i.p.), significantly increased the pulse number threshold and decreased the primary afterdischarge duration compared to those in vehicle-treated animals. In contrast, higher doses (6 or 20 mg/kg, i.p.) of fluoxetine did not significantly alter any of the seizure parameters measured. These results suggest that, in this model, stimulation of 5-HT_1A, 5-HT_2A,C and 5-HT₃ receptors does not alter seizure threshold or severity and that the blockade of 5-HT uptake produced by a low dose of fluoxetine appears to increase seizure threshold and decrease seizure severity.     

Characterization of a novel effect of serotonin 5-HT1A and 5-HT2A receptors: increasing cGMP levels in rat frontal cortex

Elucidating the mechanisms of action of hallucinogens has become an increasingly important area of research as their abuse has grown in recent years. Although serotonin receptors appear to play a role in the behavioral effects of the phenethylamine and indoleamine hallucinogens, the signaling pathways activated by these agents are unclear. Here it is shown that administration of serotonin (5-hydroxytryptamine, 5-HT) increased cyclic guanosine monophosphate (cGMP) production in frontal cortical slices of rat brain. The effect of 5-HT was greater than that of N-methyl-D-aspartate (NMDA), a stimulant of cGMP formation in the central nervous system. The 5-HT_2A/2C receptor phenethylamine agonist, 2,5-dimethoxy-4-methylamphetamine (DOM), increased cGMP content in the slices. Additionally 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), a 5-_HT1A/7 receptor agonist also increased cGMP production. Stimulation of cGMP formation by DOM was prevented by a 5-HT_2A/2C receptor antagonist, pirenperone, as well as by a 5-HT_2A receptor selective antagonist, MDL100907. A 5-HT_2C receptor antagonist, SB242084, did not block the effect of DOM. Stimulation of cGMP production by DPAT was blocked by the 5-HT_1A receptor antagonist, WAY100635. Stimulation of cGMP formation by serotonin could be prevented by pirenperone orWAY100635. In summary, activation of serotonin 5-HT_1A and 5-HT_2A receptors increase brain cGMP levels.     

Serotonin receptors and their ligands: A lack of selective agents

Four major families of serotonin (5-hydroxytryptamine; 5-HT) receptors have been identified: 5-HT₁, 5-HT₂, 5-HT₃ and 5-HT₄. At this time, there is a general consensus that the 5-HT₁ family can be further subdivided into 5-HT_1A, 5-HT_1B, 5-HT_1C, 5-HT_1D, and 5-HT_1P subpopulations. In addition, there are several other populations of less well-defined 5-HT receptors. The purpose of this presentation is to discuss 5-HT receptor nomenclature and the agents that are commonly used to investigate each receptor population in as much as it will serve to provide background for the remainder of the symposium. There is presently available an abundance of serotonergic agents; however, these agents are only semiselective, and none can be considered truly selective for a particular population of 5-HT receptors. As useful as these agents have been for the identification and characterization of 5-HT receptors, there remains a need for the development of new, more selective ligands.     

Differential effects of methamphetamine and cocaine on behavior and extracellular levels of dopamine and 3,4-dihydroxyphenylalanine in the nucleus accumbens of conscious rats

The stress-induced hyperthermia procedure, in which effects of drugs on basal (T₁) and stress-induced body temperature (T₂) are measured, predicts anxiolytic drug effect. Serotonergic drugs alter these responses and here, we studied the role of 5-HT_1A receptors in stress-induced hyperthermia by using 5-HT_1A receptor knockout mice. Three strains (129/Sv, Swiss Webster and C57Bl6) were used because genetic background can significantly modulate the null phenotype. We found that GABA-ergic drugs with an anxiolytic profile and stimulate ₂ subunit containing GABA_A receptors, including diazepam and L838,417, result in reduced ΔT (ΔT = T₂ − T₁). The ₁ subunit containing GABA_A receptor was found to be primarily involved in regulation of basal body temperature T₁ and its stimulation can induce hypothermia. In addition, stimulation of 5-HT_1A receptors by buspirone results in a reduced ΔT, while stimulation of 5-HT₇ receptors primarily results in hypothermia. The null mutation of 5-HT_1A receptors resulted in differences in drug-sensitivity that was further modulated by the genetic background. In particular, the null mutation on the SW and C57Bl6 backgrounds resulted in differential diazepam/L838,417 and 5-CT responses respectively. This indicates an interaction between the 5-HT_1A receptor and genetic background and demonstrates the importance of selecting the background strain in a receptor knockout model.     

5-HT3 receptors augment neuronal, cholinergic contractions in guinea pig trachea European Journal of Pharmacology, 234 (1993) 109–112

Serotonin (5-HT) and the selective 5-HT₃ receptor agonist, 2-methyl-5-hydroxytryptamine enhanced electrical field stimulated contractions of the isolated guinea pig trachea. 5-HT (EC₅₀ = 3.5 μM) was twice as potent as 2-methyl-5-hydroxytryptamine (EC₅₀ = 7.4 μM). The effects of 5-HT and 2-methyl-5-hydroxytryptamine were antagonized by the selective 5-HT₃ receptor antagonist, zacopride (apparent pA₂ = 7.60 against 2-methyl-5-hydroxytryptamine). 2-Methyl-5-hydroxytryptamine (10 μM) had no effect on contractile responses to exogenous acetylcholine. Furthermore, the increase in electrical field stimulated contraction by 2-methyl-5-hydroxytryptamine was unchanged by hexamethonium (100 μM) but contractions were blocked by atropine (1 μM). These results suggest that excitatory 5-HT₃ receptors exist on postganglionic cholinergic nerves in the isolated guinea pig trachea.     

A distinct distribution of functional presynaptic 5-HT receptor subtypes on GABAergic nerve terminals projecting to single hippocampal CA1 pyramidal neurons

5-HT is known to modify the excitability of GABAergic interneurons projecting to hippocampal CA1 neurons. In this study we investigate the presence and functionally characterize the 5-HT receptor subtypes found on the presynaptic nerve terminals of these GABAergic neurons. Using conventional whole-cell patch recording, we confirmed that the 5-HT_1A agonist, 8-hydroxy-2-dipropylaminotetralin, presynaptically decreased electrically evoked GABA release while the 5-HT₃ agonist, m-chlorophenylbiguanide (mCPBG), presynaptically facilitated release. Using the ‘synaptic bouton preparation’, where CA1 neurons are acutely isolated with functional nerve terminals/boutons remaining adherent, we next showed that these receptor subtypes are found presynaptically. We next used the technique of focal stimulation of a single bouton in this preparation to further investigate the distribution of these 5-HT receptor subtypes. We found that all boutons contained inhibitory 5-HT_1A receptors while a subset of boutons showed both 5-HT_1A and excitatory 5-HT₃ receptors. No boutons were detected which contained only 5-HT₃ receptors. Our studies show that presynaptic 5-HT receptor subtypes are found presynaptically and are not uniformly distributed. This provides another potential mechanism whereby 5-HT can modulate GABA release and hence the excitability of hippocampal neurons.     

Effect of quinine on autoreceptor-regulated serotonin release in the rat hippocampus

The involvement of K⁺ channels in the autoregulation of terminal serotonin (5-hydroxytryptamine, 5-HT) release was investigated by microdialysis in the hippocampus of conscious rats. Extracellular 5-HT was increased concentration-dependently by the K⁺ channel blocker quinine (10, 100 and 1000 μM in perfusate), and tetrodotoxin (10 μM) but not fluoxetine (5 μM) exerted a partially attenuating influence. The 5-HT_1/2/6 receptor antagonist methiothepin (50 μM) increased dialysate 5-HT, most likely through 5-HT_1B autoreceptors tonically activated in the hippocampus of awake rats as opposed to the previously reported lack of effect 5-HT_1B autoreceptor blockade in anesthetized rats. The effect of methiothepin was greatly reduced by preperfusion with quinine (100 μM), consonant with a role for quinine-sensitive K⁺ channels in the autoregulation of 5-HT release in the hippocampus by 5-HT receptor antagonism. In contrast, the reduction in dialysate 5-HT induced by the 5-HT₁ receptor agonist RU 24969 (1 μM), in the presence of fluoxetine (5 μM), persisted in the co-presence of quinine, consonant with the involvement of (extrasynaptic?) 5-HT autoreceptors not coupled with quinine-sensitive K⁺ channels.     

The effects of acute and repeated administration of T3 to mice on 5-HT1 and 5-HT2 function in the brain and its influence on the actions of repeated electroconvulsive shock

The effects of the administration of L-triiodothyronine (T₃) On the function of 5-HT in the CNS and its influence on the actions of electroconvulsive shock have been examined in mice. A single injection of T₃ (100 μg/kg) had no effect 24 hr later on either 5-HT_1A-mediated hypothermia, induced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.5 mg/kg) or the 5-HT_1B-mediated locomotor response to 5-methoxy-3-(l,2,3,6-tetrahydropyridin-4-yl) 1-H-indole (RU 24969; 50 ng i.c.v.). This treatment increased 5-HT₂-induced head-twitches, produced by 5-methoxy-N,N'-dimethyltryptamine (5-MeODMT; 2 mg/kg), but did not alter 5-HT₂ receptors in the frontal cortex, suggesting that this potentiation was mediated indirectly through a modulatory neurotransmitter. One injection of T₃ had no effect on the concentrations of 5-HT in the forebrain or mid/hindbrain, but increased 5-HIAA in the latter region. Daily injections of T₃ for 10 days attenuated the responses to both 8-OH-DPAT and RU 24969. Furthermore, 5-MeODMT-induced head-twitches returned to control values and this was accompanied by a 10% decrease in 5-HT₂ receptors in the cortex. Repeated administration of T₃ increased levels of 5-HT in mid/hindbrain and concentrations of 5-HIAA both here and in forebrain. Hence, treatment with T₃ attenuated the function of 5-HT_1A and 5-HT_1B receptors, but increased 5-HT₂-mediated responses, although the time-courses for these effects were different. Triiodothyronine also enhanced the synthesis and turnover of 5-HT in the brain of the mouse. Repeated electroconvulsive shock (90 V, 1 sec) decreased the hypothermia induced by 8-OH-DPAT. However, 5-MeODMT-induced head-twitches were enhanced by acute and repeated electroconvulsive shock. Administration of T₃ together with electroconvulsive shock did not alter the effects of electroconvulsive shock on 5-HT_1A-mediated hypothermia, but markedly potentiated its actions on 5-HT₂-mediated responses. These findings provide possible pharmacological evidence for the suggested antidepressant effects of T₃ and the potentiation of antidepressant therapy by this thyroid hormone.     

Pharmacological characterization of serotonin receptor subtypes involved in vasopressin and plasma renin activity responses to serotonin agonists

Serotonergic drugs with 5-HT₂ receptor agonist properties have been suggested to increase plasma vasopressin concentration, blood pressure (BP) and plasma renin activity (PRA). To study whether these actions are mediated by the same or different receptors, we used three potent 5-HT agonists with different structures and receptor binding profiles. All drugs were administered i.v. to conscious, unrestrained rats. The selective agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), which has high affinity for 5-HT₂ receptors, caused marked increases in BP and PRA but no change in plasma vasopressin concentrations. The 5-HT_1C agonist, m-chlorophenylpiperazine (m-CPP), which also binds to other 5-HT receptors, caused moderate increases in BP and PRA and significantly elevated plasma vasopressin concentrations. The 5-HT_1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), did not increase any of these parameters. BP and PRA elevations paralleled each other after all drugs, while vasopressin responses were clearly different. Vasopressin responses to m-CPP were entirely antagonised by the 5-HT₁/5-HT₂ antagonist metergoline, partially by the 5-HT₂/5-HT_1C antagonists ritanserin and LY 53857, but not by the 5-HT₂ antagonist ketanserin. Ritanserin, LY53857 and ketanserin all very effectively blocked BP responses to m-CPP. These findings suggest that BP and PRA but not vasopressin responses are mediated by 5-HT₂ receptors. Vasopressin secretion is mediated by 5-HT₁ receptors, most likely by 5-HT_1C receptors.