Sound-induced seizures in serotonin 5-HT2c receptor mutant mice

OBJECTIVES: In previous University of California, San Diego (UCSD) studies, nocturnal illumination shortened menstrual cycles that were longer than 33 days. The studies reported here extend the previous findings, confining the illumination to the sleep period. DESIGN: Two light levels (235 to 250 lux and less than 1 lux) and 2 modes of light delivery (lighted sleep mask and bedside lamp) were tested. RESULTS: 235 to 250 lux treatment cycle lengths were significantly shorter than baseline, but not significantly shorter than the less than 1 lux treatment cycle lengths. Subjective reports of sleep disturbance were greater with the 235 to 250 lux treatment, but there was no significant difference in overall quality of sleep between the two light levels. CONCLUSIONS: The current data alone do not exclude spontaneous remission or suggestion, but our previous studies demonstrated significant contrasts between 235 to 250 lux and less than 1 lux light levels. This study suggests that treatment may be effective when confined to the sleep period, and that light masks, which do not disturb bed partners, may be used in place of bedside lamps.     

Spiperone: influence of spiro ring substituents on 5-HT2A serotonin receptor binding

Spiperone (1) is a widely used pharmacological tool that acts as a potent dopamine D2, serotonin 5-HT1A, and serotonin 5-HT2A antagonist. Although spiperone also binds at 5-HT2C receptors, it is one of the very few agents that display some (ca. 1000-fold) binding selectivity for 5-HT2A versus 5-HT2C receptors and, hence, might serve as a useful template for the development of novel 5-HT2A antagonists if the impact of its various substituent groups on binding was known. In the present investigation we focused on the 1, 3,8-triazaspiro[4.5]decanone portion of spiperone and found that replacement of the N1-phenyl group with a methyl group only slightly decreased affinity for cloned rat 5-HT2A receptors. However, N1-methyl derivatives displayed significantly reduced affinity for 5-HT1A, 5-HT2C, and dopamine D2 receptors. Several representative examples were shown to behave as 5-HT2 antagonists. As such, N1-alkyl analogues of spiperone may afford entry into a novel series of 5-HT2A-selective antagonists.     

Receptor subtype and density determine the coupling repertoire of the 5-HT2 receptor subfamily

The 5-Hydroxytryptamine (5-HT)2C receptor (originally known as the 5-HT1C receptor) is a member of the 5-HT2 subfamily of G protein coupled receptors, which is known to couple to phospholipase C. Within the 5-HT2 subfamily, only the 5-HT2C receptor also coupled to inhibition of forskolin-stimulated cAMP production when expressed at high density (12 pmol/mg membrane protein) in stably transformed AV12 cells. The 5-HT2C receptor coupled with high efficacy to both phospholipase C as measured by IP3 (inositol 1,4,5-trisphosphate) production and to inhibition of forskolin-stimulated cAMP production (EC50 = 2.98 nM +/- 0.9 and IC50 = 47.99 nM +/- 10.25 respectively). The 5-HT2A and 5-HT2B receptors, while coupling to phospholipase C with high affinity (EC50s of 19.24 nM +/- 6.44 and 1.24 nM +/- 0.136 respectively), did not decrease adenylyl cyclase activity. The 5-HT2C receptor actions in both systems showed the expected pharmacology for the 5-HT2C receptor, e.g., mesulergine antagonized the effects of 5-HT and spiperone did not. Preincubation of cells with PTX showed that the G protein coupling of the 5-HT2C receptor to phospholipase C is PTX insensitive, while the G protein coupling to inhibition of adenylyl cyclase is PTX sensitive, even to concentrations as low as 20 ng/ml of PTX. PTX pretreatment of the 5-HT2C bearing cells also unmasked a small stimulatory effect on adenylyl cyclase. When expressed at low density the 5-HT2C receptor potentiated forskolin-stimulated cAMP production by 2 fold while still maintaining its ability to enhance PI hydrolysis. A more modest potentiation of cAMP production was noted with low density expression of the 5-HT2B receptor. Thus the ability of the 5-HT2C receptor to interact with several effectors through at least two different G proteins is, in part, receptor subtype specific but also influenced by receptor density.     

Perturbed dentate gyrus function in serotonin 5-HT2C receptor mutant mice

Serotonin systems have been implicated in the regulation of hippocampal function. Serotonin 5-HT2C receptors are widely expressed throughout the hippocampal formation, and these receptors have been proposed to modulate synaptic plasticity in the visual cortex. To assess the contribution of 5-HT2C receptors to the serotonergic regulation of hippocampal function, mice with a targeted 5-HT2C-receptor gene mutation were examined. An examination of long-term potentiation at each of four principal regions of the hippocampal formation revealed a selective impairment restricted to medial perforant path-dentate gyrus synapses of mutant mice. This deficit was accompanied by abnormal performance in behavioral assays associated with dentate gyrus function. 5-HT2C receptor mutants exhibited abnormal performance in the Morris water maze assay of spatial learning and reduced aversion to a novel environment. These deficits were selective and were not associated with a generalized learning deficit or with an impairment in the discrimination of spatial context. These results indicate that a genetic perturbation of serotonin receptor function can modulate dentate gyrus plasticity and that plasticity in this structure may contribute to neural mechanisms underlying hippocampus-dependent behaviors.     

Linkage of antisocial alcoholism to the serotonin 5-HT1B receptor gene in 2 populations

We examined the effects of repeated administration of (S)-alpha-fluoromethylhistidine (FMH), a specific inhibitor of histidine decarboxylase (HDC), on radial maze performance and brain contents of histamine and amino acids in rats. By daily subcutaneous (s.c.) administration of FMH (100 mg/kg), rats showed significant enhancement of a radial maze performance without changes in locomotion. Six days after FMH treatment, the histamine levels both in the cerebral cortex and diencephalon decreased significantly. However, the glutamate and glycine levels significantly increased in the cerebral cortex and hippocampus. These results suggest that FMH enhances the acquisition phase of radial maze study with the increases in glutamate and glycine levels in the cerebral cortex and hippocampus of rats.     

A receptor subtype involved in neuropeptide-Y-induced food intake

Neuropeptide Y (NPY) is a powerful stimulant of food intake and is proposed to activate a hypothalamic 'feeding' receptor distinct from previously cloned Y-type receptors. This receptor was first suggested to explain a feeding response to NPY and related peptides, including NPY2-36, that differed from their activities at the Y1 receptor. Here we report the expression cloning of a novel Y-type receptor from rat hypothalamus, which we name Y5. The complementary DNA encodes a 456-amino-acid protein with less than 35% overall identity to known Y-type receptors. The messenger RNA is found primarily in the central nervous system, including the paraventricular nucleus of the hypothalamus. The extent to which selected peptides can inhibit adenylate cyclase through the Y5 receptor and stimulate food intake in rats correspond well. Our data support the idea that the Y5 receptor is the postulated 'feeding' receptor, and may provide a new method for the study and treatment of obesity and eating disorders.     

Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice

The brain serotonin (5-hydroxytryptamine; 5-HT) system is a powerful modulator of emotional processes and a target of medications used in the treatment of psychiatric disorders. To evaluate the contribution of serotonin 5-HT1A receptors to the regulation of these processes, we have used gene-targeting technology to generate 5-HT1A receptor-mutant mice. These animals lack functional 5-HT1A receptors as indicated by receptor autoradiography and by resistance to the hypothermic effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Homozygous mutants display a consistent pattern of responses indicative of elevated anxiety levels in open-field, elevated-zero maze, and novel-object assays. Moreover, they exhibit antidepressant-like responses in a tail-suspension assay. These results indicate that the targeted disruption of the 5-HT1A receptor gene leads to heritable perturbations in the serotonergic regulation of emotional state. 5-HT1A receptor-null mutant mice have potential as a model for investigating mechanisms through which serotonergic systems modulate affective state and mediate the actions of psychiatric drugs.     

Differential effects of serotonin (5-HT) lesions and synthesis blockade on neuropeptide-Y immunoreactivity and 5-HT1A, 5-HT1B/1D and 5-HT2A/2C receptor binding sites in the rat cerebral cortex

The present study was aimed at comparing the effects of serotonin (5-HT) synthesis blockade using chronic administration of p-chlorophenylalanine (PCPA) and 5,7-dihydroxytryptamine injections of variable volume (3 vs. 6 microl) on the density of NPY immunoreactive (Ir) neurons and binding of [3H]8-OH-DPAT, S-CM-G[125I]TNH2 and [125I]DOI to 5-HT1A, 5-HT1B/1D, and 5-HT2A/2C receptors in rat cortical regions. Three weeks after large but partial (89% depletion in 5-HT tissue concentration) lesions of 5-HT neurons no changes in neither NPY immunoreactivity nor 5-HT receptor binding were detected. The complete 5,7-DHT lesions produced increases in the number of NPY-Ir neurons in the upper regions of the cingular (134%), frontal (140%) and parietal cortex (48%) and corresponding decreases in 5-HT2A/2C binding (16-26%). No changes in 5-HT1A and 5-HT1B/1D binding were observed after lesions of this kind. After PCPA treatment, decreases in NPY-Ir neurons density (22-40%) and increases in 5-HT1A and 5-HT1B/1D receptor binding sites (20-50%) were distributed in both upper and deeper cortical regions. The lack of effect of the partial lesion suggests that spared 5-HT neurons may exert compensatory mechanisms up to a large extent. The changes in NPY immunoreactivity and 5-HT2A/2C binding detected in the upper regions of the cortex after complete 5-HT lesions probably result from local cellular rearrangements, whereas blocking 5-HT synthesis has more widespread influence on NPY neurons and on 5-HT1A and 5-HT1B/1D receptor subtypes. Moreover, decreases in DOPAC concentrations detected only after complete lesions suggest that the involvement of catecholaminergic transmission may also differentiate 5,7-DHT and PCPA treatments. Altogether, these data suggest that different receptor subtypes might be involved in 5-HT-NPY relationships.     

Involvement of the 5-HT1A subtype receptor in the neonatal organization of agonistic behaviour in the rat

5-hydroxytryptamine (5-HT) interacts with testosterone (T) in the development of a number of neuronal systems controlling sexually dimorphic adult behaviours. In this report, we investigated this interaction on the organization of agonistic behaviour in males, females, androgenized females (250 micrograms/pup of T proprionate on the day of birth), and males castrated on the day of birth. We have shown previously that manipulating 5-HT2 activity over the 2nd week of life modulates adult agonistic behaviour, depending on genetic sex and the presence of T. In this report, we investigated the effects seen in adulthood of a 5-HT1A agonist [8-OH-DPAT, 0.25 mg/kg, intraperitoneally (i.p.)] and antagonist (WAY100135, 0.25 mg/kg, i.p.) given over days 8-16 postpartum. The test for agonistic behaviour was carried out in a neutral territory against a matched conspecific, and introductory, offensive and defensive activities were note. Results show that neonatal administration of the 5-HT1A antagonist WAY100135 increases introductory activity and defense in the presence of neonatal T, independent of genetic sex, because these effects were seen in sham-castrated males and androgenized females. Offence followed a similar pattern, in that it was increased by WAY100135, but only in males. In the case of defence, the effects of the antagonist were reinforced by the action of the agonist (8-OH-DPAT) in both males and females, indicating an inhibitory role of 5-HT1A perinatal activity on defence in the presence of malelike levels of circulating T and a facilitatory role when levels of T are low or negligible. These findings indicate that 5-HT1A activity is involved in the development of agonistic behaviour and the effects are influenced by T. The results also show that the offensive and defensive facets of agonistic activity are controlled differently.     

5-HT2A and 5-HT2C receptor polymorphisms and psychopathology in late onset Alzheimer's disease

The psychopathology of Alzheimer's disease (AD) is varied and includes both behavioural and psychological symptoms. Behavioural and psychological symptoms are common and contribute to the difficulties experienced by carers. However, the mechanism whereby these symptoms occur in some individuals with AD is not understood. We hypothesized that common genetic polymorphisms in neurotransmitter systems are risk factors for these symptoms in the course of AD. A total of 211 subjects from a population-based prospective study of psychopathology within late-onset AD were genotyped for the 5-HT2A receptor polymorphism 102-T/C and the 5-HT2C receptor polymorphism Cys23Ser. Associations were found between the presence of the C102 allele and the presence of visual (Fisher's exact test, one-tailed, P = 0.003) and auditory hallucinations (Fisher's exact test, one-tailed, P = 0.004) and between the presence of the Ser23 allele and visual hallucinations (chi2 = 7.5, df = 1, P = 0.006) (P = 0.03, 0.04 and 0.06, respectively, after Bonferroni correction). In addition, there was an association between the Cys23Ser polymorphism and hyperphagia (chi2 = 6.7, df = 2, P = 0.03) (P = 0.3 after Bonferroni correction). We conclude that common 5-HT2A and 5-HT2C genetic polymorphisms previously showing only weak associations with psychotic illness are associated with psychotic symptoms in AD but are clinically silent until the onset of the neurodegenerative process.     

Animal models for studying serotonin (5-HT) receptor subtypes: relationship to 5-HT system pathologies

Despite more than 30 years of intensive research, the specific role of serotonin (5-HT) receptor subtypes in animal models of "anxiety" still remains unclear. The present study focused on the particular role of 5-HT1A receptor subtype in aversive learning, i.e., the passive avoidance (PA) task in the rat. Taken together, the data strongly suggest that: (1) 5-HT1A receptor but not 5-HT2A receptors play a crucial role in PA; (2) the postsynaptic but not presynaptic 5-HT1A receptors are mainly involved in the regulation of PA; (3) 5-HT1A receptors appear to be directly involved both in acquisition and retrieval but not in the consolidation of PA; and (4) besides the "prototypical" 5-HT1A receptor subtype, an additional and yet-unidentified 5-HT receptor subtype seems to play an important modulatory role in PA.     

Stimulation of type 1 and type 8 Ca2+/calmodulin-sensitive adenylyl cyclases by the Gs-coupled 5-hydroxytryptamine subtype 5-HT7A receptor

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important regulatory role in developing and adult nervous systems. With the exception of the 5-HT3 receptor, all of the cloned serotonin receptors belong to the G protein-coupled receptor superfamily. Subtypes 5-HT6 and 5-HT7 couple to stimulation of adenylyl cyclases through Gs and display high affinities for antipsychotic and antidepressant drugs. In the brain, mRNA for 5-HT6 is found at high levels in the hippocampus, striatum, and nucleus accumbens. 5-HT7 mRNA is most abundant in the hippocampus, neocortex, and hypothalamus. To better understand how serotonin might control cAMP levels in the brain, we coexpressed 5-HT6 or 5-HT7A receptors with specific isoforms of adenylyl cyclase in HEK 293 cells. The 5-HT6 receptor functioned as a typical Gs-coupled receptor in that it stimulated AC5, a Gs-sensitive adenylyl cyclase, but not AC1 or AC8, calmodulin (CaM)-stimulated adenylyl cyclases that are not activated by Gs-coupled receptors in vivo. Surprisingly, serotonin activation of 5-HT7A stimulated AC1 and AC8 by increasing intracellular Ca2+. 5-HT also increased intracellular Ca2+ in primary neuron cultures. These data define a novel mechanism for the regulation of intracellular cAMP by serotonin.     

Long-term sequential determination of behavioral ontogeny of 5-HT1A and 5-HT2 receptor functions in the rat

Activation of 5-hydroxytryptamine1A (5-HT1A) receptors in rats produces hypothermia and a number of behaviors [hindleg abduction (HLA), lateral head-weaving (LHW), forepaw treading (FPT), flat body posture (FBP), rollover (RO), tremor (T), and straub tail (ST)] known collectively as the serotonin syndrome (SS). Stimulation of 5-HT2A receptors produces wet-dog shakes (WDS), whereas 5-HT2C sites induce back muscle contraction (BMC). We investigated the functional ontogeny of the cited receptors in rat pups on postnatal days (PD) 7, 14, 18, 22, 28, 35, 60, and 120 by using (1) the 5-HT1A agonist 8-hydroxy-2-dipropylaminotetralin (0, 1.25, and 5 mg/kg) to induce the SS and hypothermia and (2) the 5-HT2A/C agonist (+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane (0, 0.5, and 4 mg/kg) to produce both WDS and BMC. The age of onset for most symptoms of SS [FBP, HLA, RO, and T] was the first week of life. They attained maximal intensities at ages 7 to 14 days, after which their maxima either reduced or dissipated to zero. Per contra, the onset of LHW and FPT required 14 to 18 days, and their maxima developed later. The onset of (+/-)-1-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropane-induced WDS occurred after PD 14, and by PD 18, it reached its maximal intensity, which persisted up to PD 60, after which it declined. The onset of BMC was evident on PD 28 and attained its maximal frequency at ages 90 to 120 days. The results show that different components of SS appear within 14 days of birth, but they mature differentially, whereas the hypothermic effect of 5-HT1A receptors remains relatively constant during aging. The times of onset and maturation of WDS were intermediate (between the second and third weeks of life), whereas BMC required 1 to 2 months for its appearance and maturation.     

Molecular modeling of serotonin, ketanserin, ritanserin and their 5-HT2C receptor interactions

Molecular modeling techniques were used to build a three-dimensional model of the rat 5-HT2C receptor, which was used to examine receptor interactions for protonated forms of serotonin, ketanserin and ritanserin. Molecular dynamics simulations which were started with the fluoro benzene moiety of ketanserin and ritanserin oriented towards the cytoplasmic side of the receptor model, produced the strongest antagonist-receptor interactions. The fluoro bezene ring(s) of the antagonists interacted strongly with aromatic residues in the receptor model, which predicts slightly different orientations and ligand-receptor interactions of ketanserin and ritanserin at a putative binding site. The model suggests that Asn333 (transmembrane helix 6) is involved in a hydrogen-bonding interaction with ketanserin, but not with ritanserin. The model also also suggests that the position corresponding to Cys362 (transmembrane helix 7) may be an important determinant for specifying 5-HT2A receptor selectivity in ketanserin binding.     

Lack of association between neuroleptic malignant syndrome and polymorphisms in the 5-HT1A and 5-HT2A receptor genes

OBJECTIVE: The molecular basis of neuroleptic malignant syndrome is unclear, but studies suggest that genetic factors are involved in its pathogenesis. Considering possible involvement of the serotonergic system in neuroleptic malignant syndrome, the authors examined the association between neuroleptic malignant syndrome and polymorphisms of the 5-HT1A and 5-HT2A receptor genes. METHOD: The authors examined the frequencies of gene polymorphisms in the 5-HT1A (Arg219Leu) and 5-HT2A (Thr25Asn and His452Tyr) receptor genes in 29 patients previously diagnosed with neuroleptic malignant syndrome, 94 neuroleptic-treated patients with schizophrenia who had no history of neuroleptic malignant syndrome, and 94 healthy comparison subjects. Polymerase chain reaction and restriction fragment length polymorphism analyses were used to screen gene mutations. RESULTS: No polymorphic allele was detected in the patients who had experienced the neuroleptic malignant syndrome. CONCLUSIONS: The authors cannot conclude that polymorphisms in the 5-HT1A and 5HT2A receptor genes are factors determining susceptibility to the neuroleptic malignant syndrome.     

Immunohistochemical localisation of the serotonin 5-HT2B receptor in mouse gut, cardiovascular system, and brain

We recently reported the cloning of a new member of the serotonin 5-HT2 family, the 5-HT2B receptor. We now report the production and characterisation of a specific antiserum directed against the C-terminal portion of the mouse 5-HT2B receptor. After affinity purification, this polyclonal antibody recognises specifically the mouse 5-HT2B receptor. Immunohistochemical analysis of cryosections from various adult mouse tissues reveals a major 5-HT2B receptor expression in stomach, intestine and pulmonary smooth muscles as well as in myocardium. Furthermore, the antiserum recognises specific areas of the mouse brain, including cerebellar Purkinje cells and their projection areas.     

Association analysis of the 5-HT2C receptor and 5-HT transporter genes in bipolar disorder

We selected 42 patients with bipolar disorder type I (BPI) and 40 healthy controls for genetic analysis of DNA polymorphisms in the serotonin receptor 2c (5-HTR2c) and serotonin transporter (5-HTT) genes. No significant associations were found in the total patient sample. However, when the individuals were divided according to gender, trends for association with both polymorphisms (P = 0.051 for 5-HTR2c and P = 0.049 for 5-HTT) in female patients were observed. These results suggest that variations in these genes may be responsible for a minor increase in susceptibility for bipolar disorder in women.     

Characterization of a mouse serotonin 5-HT3 receptor purified from mammalian cells

A serotonin 5-HT3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one-step purification yielding approximately 50% of the histidine-tagged 5-HT3 receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild-type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of approximately 5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5-HT3 receptor is a pentameric homooligomer. The secondary structure of the 5-HT3 receptor as determined by circular dichroism appeared to consist of mainly alpha-helices (50%) and beta-strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.     

Sigma 1 receptor subtype is involved in the facilitation of cortical dopaminergic transmission in the rat brain

Our previous studies have shown that three sigma (sigma) receptor ligands, (+)-N-allylnormetazocine ((+)-SKF-10,047), (+/-)-pentazocine and 1,3-di(2-tolyl)guanidine (DTG) differently regulated the dopamine (DA) transmission in the rat brain. In the present study, we attempted to clarify the role of sigma 1 receptor subtype in the regulation of DA transmission using a novel and selective sigma 1 receptor agonist, 1-(3,4-dimethoxyphenethyl)-4(3-phenylpropyl)piperazine dihydrochloride (SA4503) in the rat brain. Acute administration of SA4503 (1.0 mg/kg, p.o.) significantly increased DA and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the rat frontal cortex, but not in the other six regions, hippocampus, striatum, midbrain, cerebellum, medulla/pons and hypothalamus. The increase of cortical DA level elicited by SA4503 was fully reversed by N,N-dipropyl-2-(4-methoxy-3-(2-phenylethoxy)phenyl)ethylamine (NE-100) (0.25 mg/kg, p.o.), a putative sigma 1 receptor antagonist. In addition, SA4503 (1.0 mg/kg, p.o.) showed an increase of cortical L-3,4-dihydroxyphenylalanine (L-DOPA) accumulation under the inhibition of dopa decarboxylase activity with m-hydrobenzylhydrazine (NSD-1015), suggesting that SA4503 has activated the cortical DA synthesis rate. These results suggest that the sigma 1 receptor subtype plays an important role in the facilitation of cortical DA transmission. In addition, this phenomenon is partially involved in the augmentation of DA synthesis rate.     

Operational characteristics of the 5-HT1-like receptors mediating external carotid vasoconstriction in vagosympathectomized dogs. Close resemblance to the 5-HT1D receptor subtype

It has recently been shown that the external carotid vasoconstrictor response to 5-HT in the dog is primarily mediated by sumatriptan-sensitive 5-HT1-like receptors; however, the fact that these receptors are not blocked by metergoline, a 5-HT1D ligand, raises questions about their possible correlation with the 5-HT1D receptor subtype. Since a number of drugs display high affinity for the 5-HT1D (GR127935) and 5-HT1F (e.g. methysergide and oxymetazoline) receptor subtypes, in this study we have used these drugs to determine whether the above vasoconstrictor 5-HT1-like receptors correlate with the 5-HT1D and/or 5-HT1F receptor subtypes. One-minute intracarotid infusions of 5-HT (0.3-30 micrograms/min), sumatriptan (1-30 micrograms/min), oxymetazoline (0.03-3 micrograms/min) and noradrenaline (0.3-3 micrograms/min) resulted in dose-dependent decreases in external carotid blood flow without changes in arterial blood pressure or heart rate. These vasoconstrictor responses remained unaltered after i.v. administration of physiological saline (0.015, 0.05 and 0.15 ml/kg; n = 4) or ritanserin (1 mg/kg; n = 5). In contrast, GR127935 (1, 3 and 10 micrograms/kg, n = 6) potently blocked the responses to 5-HT (unmasking a dose-dependent vasodilator component) and sumatriptan without affecting those to oxymetazoline or noradrenaline. Interestingly, methysergide (10, 30 and 100 micrograms/kg, n = 5) also blocked the vasoconstrictor responses to 5-HT and sumatriptan, but unlike GR127935, did not revert the vasoconstrictor response to 5-HT; the responses to oxymetazoline remained unaffected, but those to noradrenaline were apparently attenuated by the highest dose. Taken together, the above findings suggest that the sumatriptan-sensitive 5-HT1-like receptors mediating canine external carotid vasoconstriction resemble 5-HT1D receptors, probably of the 5-HT1D beta subtype on the basis of the resistance to blockade by ritanserin. The pharmacological profile of these receptors could be similar (bovine and human cerebral arteries, porcine carotid arteriovenous anastomoses and human coronary arteries) to other putative 5-HT1D receptors mediating vascular responses.