Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues.

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.

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.

Modulating the rate and rhythmicity of perceptual rivalry alternations with the mixed 5-HT2A and 5-HT1A agonist psilocybin

Binocular rivalry occurs when different images are presented simultaneously to corresponding points within the left and right eyes. Under these conditions, the observer's perception will alternate between the two perceptual alternatives. Motivated by the reported link between the rate of perceptual alternations, symptoms of psychosis and an incidental observation that the rhythmicity of perceptual alternations during binocular rivalry was greatly increased 10 h after the consumption of LSD, this study aimed to investigate the pharmacology underlying binocular rivalry and to explore the connection between the timing of perceptual switching and psychosis. Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine, PY) was chosen for the study because, like LSD, it is known to act as an agonist at serotonin (5-HT)(1A) and 5-HT2A receptors and to produce an altered state sometimes marked by psychosis-like symptoms. A total of 12 healthy human volunteers were tested under placebo, low-dose ( 115 mg/kg) and high-dose ( 250 mg/kg) PY conditions. In line with predictions, under both low- and high-dose conditions, the results show that at 90 min postadministration ( the peak of drug action), rate and rhythmicity of perceptual alternations were significantly reduced from placebo levels. Following the 90 min testing period, the perceptual switch rate successively increased, with some individuals showing increases well beyond pretest levels at the final testing, 360 min postadministration. However, as some subjects had still not returned to pretest levels by this time, the mean phase duration at 360 min was not found to differ significantly from placebo. Reflecting the drug-induced changes in rivalry phase durations, subjects showed clear changes in psychological state as indexed by the 5D-ASC ( altered states of consciousness) rating scales. This study suggests the involvement of serotonergic pathways in binocular rivalry and supports the previously proposed role of a brainstem oscillator in perceptual rivalry alternations and symptoms of psychosis.

The heteromeric 5-HT3A/B receptor:the effect of 5-HT3B subunits on receptor structure and ligand recognition

The 5-HT3 receptors are members of the cys-loop family of ligand-gated ion channels. Two functional subtypes are known, the homomeric 5HT3A and the heteromeric 5HT3A/B receptors, which exhibit distinct biophysical characteristics but are difficult to differentiate pharmacologically. Atomic force microscopy has been used to determine the stoichiometry and architecture of the heteromeric 5HT3A/B receptor. Each subunit was engineered to express a unique C-terminal epitope tag, together with six sequential histidine residues to facilitate nickel affinity purification. The 5-HT3 receptors, ectopically expressed in HEK293 cells, were solubilised, purified and decorated with antibodies to the subunit specific epitope tags. Imaging of individual receptors by atomic force microscopy revealed a pentameric arrangement of subunits in the order BBABA, reading anti-clockwise when viewed from the extracellular face. Homology models for the heteromeric receptor were then constructed using both the electron microscopic structure of the nicotinic acetylcholine receptor, from Torpedo marmorata, and the X-ray crystallographic structure of the soluble acetylcholine binding protein, from Lymnaea stagnalis, as templates. These homology models were used, together with equivalent models constructed for the homomeric receptor, to interpret mutagenesis experiments designed to explore the minimal recognition differences of both the natural agonist, 5-HT, and the competitive antagonist, granisetron, for the two human receptor subtypes. The results of this work revealed that the 5-HT3B subunit residues within the ligand binding site, for both the agonist and antagonist, are accommodating to conservative mutations. They are consistent with the view that the 5-HT3A subunit provides the principal and the 5-HT38 subunit the complementary recognition interactions at the binding interface.

Drug action on anxiety models with special reference to serotonergic mechanisms

A study has been made of drugs acting at 5-HT receptors on animal models of anxiety. An elevated X-maze was used as a model of anxiety for rats and the actions of various ligands for the 5-HT receptor, and its subtypes, were examined in this model. 5-HT agonists, with varying affinities for the 5-HT receptor subtypes, were demonstrated to have anxiogenic-like activity. The 5-HT2 receptor antagonists ritanserin and ketanserin exhibited an anxiolytic-like profile. The new putatuve anxiolytics ipsapirone and buspirone, which are believed to be selective for 5-HT1 receptors, were also examined. The former had an anxiolytic profile whilst the latter was without effect. Antagonism studies showed the anxiogenic response to 8-hydroxy-2-(Di-n-propylamino)tetralin (8-OH-DPAT) to be antagonised by ipsapirone, pindolol, alprenolol and para-chlorophenylalanine, but not by diazepam, ritanserin, metoprolol, ICI118,551 or buspirone. To confirm some of the results obtained in the elevated X-maze the Social Interaction Test of anxiety was used. Results in this test mirrored the effects seen with the 5-HT agonists, ipsapirone and pindolol, whilst the 5-HT2 receptor antagonists were without effect. Studies using operant conflict models of anxiety produced marginal and varying results which appear to be in agreement with recent criticisms of such models. Finally, lesions of the dorsal raphe nucleus (DRN) were performed in order to investigate the mechanisms involved in the production of the anxiogenic response to 8-OH-DPAT. Overall the results lend support to the involvement of 5-HT, and more precisely 5-HT1, receptors in the manifestation of anxiety in such animal models.

An Ins(1,4,5)P3 receptor in Paramecium is associated with the osmoregulatory system

In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.

Galanin and galanin fragment 1-15 modulate antidepressant responses by targeting 5-HT1AR-GALR heteroreceptor complexes of the ascending midbrain serotonin pathways.

Mood disorders, including depression and anxiety, are among the most prevalent mental illnesses with high socioeconomic impact. Although the underlying mechanisms have not yet been clearly defined in the last decade the importance of the role of neuropeptides, including Galanin (GAL), and/or their receptors in the treatment of stress-related mood disorders is becoming increasingly apparent. GAL is involved in mood regulation, including depression-related and anxiety-like behaviors. Activation of GALR1 and GALR3 receptors results in a depression like behavior while stimulation of GALR2 receptor leads to anti-depressant-like effects. Moreover, GAL modulates 5-HT1A receptors (5-HT1AR), a key receptor in depression at autoreceptor and postsynaptic level in the brain. This interaction can in part be due to the existence of GALR1-5-HT1AR heteroreceptor complexes in discrete brain regions [1]. Not only GAL but also the N-terminal fragments like GAL(1-15) are active in the Central Nervous System [2, 3]. Recently, we described that GAL(1-15) induces strong depression-related and anxiogenic-like effects in rats, and these effects were significantly stronger than the ones induced by GAL [4]. The GALR1-GALR2 heteroreceptor complexes in the dorsal hippocampus and especially in the dorsal raphe (DR), areas rich in GAL(1-15) binding sites [5] were involved in these effects [4, 6] and demonstrated also in cellular models. In the present study, we have analyzed the ability of GAL(1-15) to modulate 5-HT1AR located at postjunctional sites and at the soma-dendritic level in rats. We have analyzed the effect of GAL(1-15) on the 5-HT1AR-mediated response in a behavioral test of depression and the involvement of the GALR2 in these effects. GAL(1-15) enhanced the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT in the forced swimming test [7]. These effects were stronger than the ones induced by GAL. The mechanism of this action involved interactions at the receptor level in the plasma membrane with changes also at the transcriptional level. Thus, GAL(1-15) affected the binding characteristics as well as the mRNA level of 5-HT1AR in the dorsal hippocampus and DR. GALR2 was involved in these effects, since the specific GALR2 antagonist M871 blocked GAL(1-15) mediated actions at the behavioral and receptor level [7]. Furthermore, the results on the proximity ligation assay (PLA) in this work suggest the existence of GALR1-GALR2-5-HT1AR heteroreceptor complexes since positive PLA were obtained for both GALR1-5-HT1AR and GALR2-5-HT1AR complexes in the DR and hippocampus. Moreover the studies on RN33B cells, where GALR1, GALR2 and 5-HT1AR exist [4], also showed PLA-positive clusters indicating the existence of GALR1-5-HT1AR and GALR2-5-HT1AR complexes in these cells [7]. In conclusion, our results indicate that GAL(1–15) enhances the antidepressant effects induced by the 5-HT1AR agonist 8-OH-DPAT probably acting on GALR1-GALR2-5-HT1AR heteroreceptor located at postjunctional sites and at the soma-dendritic level. The development of new drugs specifically targeting these heteroreceptor complexes may offer a novel strategy for treatment of depression. This work has been supported by Junta de Andalucia CVI646 1. Borroto-Escuela, D.O., et al., Galanin receptor-1 modulates 5-hydroxtryptamine-1A signaling via heterodimerization. Biochem Biophys Res Commun, 2010. 393(4): p. 767-72. 2. Hedlund, P.B. and K. Fuxe, Galanin and 5-HT1A receptor interactions as an integrative mechanism in 5-HT neurotransmission in the brain. Ann N Y Acad Sci, 1996. 780: p. 193-212. 3. Diaz-Cabiale, Z., et al., Neurochemical modulation of central cardiovascular control: the integrative role of galanin. EXS, 2010. 102: p. 113-31. 4. Millon, C., et al., A role for galanin N-terminal fragment (1-15) in anxiety- and depression-related behaviors in rats. Int J Neuropsychopharmacol, 2015. 18(3). 5. Hedlund, P.B., N. Yanaihara, and K. Fuxe, Evidence for specific N-terminal galanin fragment binding sites in the rat brain. Eur J Pharmacol, 1992. 224(2-3): p. 203-5. 6. Borroto-Escuela, D.O., et al., Preferential activation by galanin 1-15 fragment of the GalR1 protomer of a GalR1-GalR2 heteroreceptor complex. Biochem Biophys Res Commun, 2014. 452(3): p. 347-53. 7. Millon, C., et al., Galanin (1-15) enhances the antidepressant effects of the 5-HT1A receptor agonist 8-OH-DPAT: involvement of the raphe-hippocampal 5-HT neuron system. Brain Struct Funct, 2016.

Cigarette smoking in young adults : the influence of the HTR2A T102C polymorphism and punishment sensitivity

Background: The C allele of a common polymorphism of the serotonin 2A receptor (HTR2A) gene, T102C, results in reduced synthesis of 5-HT2A receptors and has been associated with current smoking status in adults. The -1438A/G polymorphism, located in the regulatory region of this gene, is in linkage disequilibrium with T102C, and the A allele is associated with increased promoter activity and with smoking in adult males. We investigated the contributions of the HTR2A gene, chronic psychological stress, and impulsivity to the prediction of cigarette smoking status and dependence in young adults. Methods: T102C and -1438A/G genotyping was conducted on 132 healthy Caucasian young adults (47 smokers) who completed self-report measures of chronic stress, depressive symptoms, impulsive personality and cigarette use. Results: A logistic regression analysis of current cigarette smoker user status, after adjusting for gender, depressive symptom severity and chronic stress, indicated that the T102C TT genotype relative to the CC genotype (OR = 7.53), and lower punishment sensitivity (OR = 0.91) were each significant predictive risk factors. However, for number of cigarettes smoked, only lower punishment sensitivity was a significant predictor (OR = 0.81). Conclusions: These data indicate the importance of the T102C polymorphism to tobacco use but not number of cigarettes smoked for Caucasian young adults. Future studies should examine whether this is explained by effects of nicotine on the serotonin system. Lower punishment sensitivity increased risk of both smoking and of greater consumption, perhaps via a reduced sensitivity to cigarette health warnings and negative physiological effects.

Remodeling of purinergic receptor-mediated Ca 2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.

Mice lacking the serotonin Htr2B receptor gene present an antipsychotic-sensitive schizophrenic-like phenotype

Impulsivity and hyperactivity share common ground with numerous mental disorders, including schizophrenia. Recently, a population-specific serotonin 2B (5-HT2B) receptor stop codon (ie, HTR2B Q20*) was reported to segregate with severely impulsive individuals, whereas 5-HT2B mutant (Htr2B−/−) mice also showed high impulsivity. Interestingly, in the same cohort, early-onset schizophrenia was more prevalent in HTR2B Q*20 carriers. However, the putative role of 5-HT2B receptor in the neurobiology of schizophrenia has never been investigated. We assessed the effects of the genetic and the pharmacological ablation of 5-HT2B receptors in mice subjected to a comprehensive series of behavioral test screenings for schizophrenic-like symptoms and investigated relevant dopaminergic and glutamatergic neurochemical alterations in the cortex and the striatum. Domains related to the positive, negative, and cognitive symptom clusters of schizophrenia were affected in Htr2B−/− mice, as shown by deficits in sensorimotor gating, in selective attention, in social interactions, and in learning and memory processes. In addition, Htr2B−/− mice presented with enhanced locomotor response to the psychostimulants dizocilpine and amphetamine, and with robust alterations in sleep architecture. Moreover, ablation of 5-HT2B receptors induced a region-selective decrease of dopamine and glutamate concentrations in the dorsal striatum. Importantly, selected schizophrenic-like phenotypes and endophenotypes were rescued by chronic haloperidol treatment. We report herein that 5-HT2B receptor deficiency confers a wide spectrum of antipsychotic-sensitive schizophrenic-like behavioral and psychopharmacological phenotypes in mice and provide first evidence for a role of 5-HT2B receptors in the neurobiology of psychotic disorders

Interazioni neurotrasmettitoriali nel meccanismo d’azione della cocaina: ruolo della serotonina e dell’adenosina

The putative 5-HT6 receptor agonist ST1936 has been shown to increase extracellular dopamine (DA) in the n.accumbens (NAc) Shell and in the medial prefrontal cortex (PFCX). These observations suggest that 5-HT6 receptors modulate DA transmission in mesolimbic and mesocortical terminal DA areas. To investigate the behavioral counterpart of this interaction I studied in rats the effect of 5-HT6 receptor blockade on cocaine stimulated overflow of DA in dialysates from the PFCX and from the NAc Shell and on cocaine i.v. selfadministration. Pretreatment with the 5-HT6 antagonist SB271046 reduced cocaine-induced increase of dialysate DA in the NAc Shell but not in the PFCX and impaired i.v. cocaine selfadministration. These suggest that 5-HT6 receptors play a role in cocaine reinforcement via their facilitatore interaction with DA projections to the NAc Shell. This 5-HT/DA interaction might provide the basis for a new pharmacotherapeutic strategy of cocaine addiction. Caffeine is one of the psychoactive substances most widely used as adulterant in illicit drugs, such as cocaine. Animal studies have demonstrated that caffeine is able to potentiate cocaine actions, although the enhancement of the cocaine reinforcing property by caffeine is less reported, and the results depend on the paradigms and experimental protocols used. In the present study I examined the ability of caffeine to enhance the motivational and rewarding properties of cocaine using the intravenous self-administration paradigm in rats. Additionally, the role of caffeine as a primer cue during extinction was evaluated. To this end, we assessed in naïve rats: 1) the ability of the combination of cocaine (0,125 mg/kg/infusion) and caffeine (0,0625 mg/kg/infusion) to maintain self-administration in fixed ratio (FR) and progressive ratio (PR) schedules of reinforcement compared with cocaine and caffeine alone; 2) the effect of caffeine in the maintenance of responding in the animals exposed to the combination of the drugs during cocaine extinction. Cocaine and the combination of cocaine and caffeine were self-administered on a FR and PR schedules of reinforcement, and the responding for the combination of the drugs was higher than cocaine alone. Caffeine was not reliably self-administered, but was able to maintain a drug-seeking behavior in rats previously exposed to cocaine plus caffeine. These findings suggest that the presence of caffeine enhances the reinforcing effects of cocaine and the motivational value of the drug. Our results highlight the role of active adulterants commonly used in illicit street drugs.

Os recetores da melatonina como novos alvos de fármacos antidepressivos

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Endothelin 1 Stimulates Ca2+-Sparks and Oscillations in Retinal Arteriolar Myocytes via IP3R and RyR-Dependent Ca2+ Release

PURPOSE:
To investigate endothelin 1 (Et1)-dependent Ca(2+)-signaling at the cellular and subcellular levels in retinal arteriolar myocytes.
METHODS:
Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using confocal laser microscopy.
RESULTS:
Basal [Ca(2+)](i), subcellular Ca(2+)-sparks, and cellular Ca(2+)-oscillations were all increased during exposure to Et1 (10 nM). Ca(2+)-spark frequency was also increased by 90% by 10 nM Et1. The increase in oscillation frequency was concentration dependent and was inhibited by the EtA receptor (Et(A)R) blocker BQ123 but not by the EtB receptor antagonist BQ788. Stimulation of Ca(2+)-oscillations by Et1 was inhibited by a phospholipase C blocker (U73122; 10 µM), two inhibitors of inositol 1,4,5-trisphosphate receptors (IP(3)Rs), xestospongin C (10 µM), 2-aminoethoxydiphenyl borate (100 µM), and tetracaine (100 µM), a blocker of ryanodine receptors (RyRs).
CONCLUSIONS:
Et1 stimulates Ca(2+)-sparks and oscillations through Et(A)Rs. The underlying mechanism involves the activation of phospholipase C and both IP(3)Rs and RyRs, suggesting crosstalk between these Ca(2+)-release channels. These findings suggest that phasic Ca(2+)-oscillations play an important role in the smooth muscle response to Et1 within the retinal microvasculature and support an excitatory, proconstrictor role for Ca(2+)-sparks in these vessels.

Pharmacological studies of 8-OH-DPAT-induced pupillary dilation in anesthetized rats

Serotonin (5-HT) receptor agonists have been reported to produce mydriasis in mice, and miosis in rabbits and humans. However, the underlying mechanisms for this action are unclear. This study was undertaken in an attempt to explore the mechanism by which 5-HT receptors are involved in the modulation of pupillary size in pentobarbital-anesthetized rats. Intravenous administration of the 5-HT receptor agonist, (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT; 0.003-3 mg/kg), elicited dose-dependent pupillary dilation, which was not affected by section of the preganglionic cervical sympathetic nerve. 8-OH-DPAT-elicited mydriatic responses were attenuated by the selective 5-HT receptor antagonist, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2- pyridinylcyclohexanecarboxamide maleate (WAY 100635; 0.3-1 mg/kg, i.v.), as well as by the selective a -adrenoceptor antagonist, (8aR,12aS,13aS)-5,8,8a,9,10,11,12,12a,13,13a-dechydro-3-methoxy-12- (ethylsulfonyl)-6H-isoquino[2,1-g][1,6]naphthyridine hydrochloride (RS 79948; 0.3 mg/kg, i.v.), but not by the selective a -adrenoceptor antagonist, prazosin (0.3 mg/kg, i.v.). Mydriatic responses elicited by the a -adrenoceptor agonist, guanabenz (0.003-0.3 mg/kg, i.v.), were not antagonized by WAY 100635 (0.3-1 mg/kg, i.v.). To determine whether central nervous system (CNS) 5-HT receptors, like a -adrenoceptors, are involved in reflex mydriasis, voltage response curves of pupillary dilation were constructed by stimulation of the sciatic nerve in anesthetized rats. WAY 100635 (1 mg/kg, i.v.) did not antagonize the evoked reflex mydriasis, which, however, was blocked by RS 79948 (0.3 mg/kg, i.v.). Taken together, these results suggest that 8-OH-DPAT produces pupillary dilation in anesthetized rats by stimulating CNS 5-HT receptors, which in turn trigger the release of norepinephrine, presumably from the locus coeruleus. The latter reduces parasympathetic neuronal tone to the iris sphincter muscle by stimulation of postsynaptic a - adrenoceptors within the Edinger-Westphal nucleus. Unlike a - adrenoceptors, 5-HT receptors in the CNS do not mediate reflex mydriasis evoked by sciatic nerve stimulation. © 2004 Elsevier B.V. All rights reserved.