5-HT1B Antagonist Research Articles

2B Determined: The Future of the Serotonin Receptor 2B in Drug Discovery.

The cardiotoxicity associated with des-ethyl-dexfenfluramine (norDF) and related agonists of the serotonin receptor 2B (5-HT2B) has solidified the receptor's place as an "antitarget" in drug discovery. Conversely, a growing body of evidence has highlighted the utility of 5-HT2B antagonists for the treatment of pulmonary arterial hypertension (PAH), valvular heart disease (VHD), and related cardiopathies. In this Perspective, we summarize the link between the clinical failure of fenfluramine-phentermine (fen-phen) and the subsequent research on the role of 5-HT2B in disease progression, as well as the development of drug-like and receptor subtype-selective 5-HT2B antagonists. Such agents represent a promising class for the treatment of PAH and VHD, but their utility has been historically understudied due to the clinical disasters associated with 5-HT2B. Herein, it is our aim to examine the current state of 5-HT2B drug discovery, with an emphasis on the receptor's role in the central nervous system (CNS) versus the periphery.

Structure activity relationships of 5-HT2B and 5-HT2C serotonin receptor antagonists: N6, C2 and 5′-Modified (N)-methanocarba-adenosine derivatives

(N)-Methanocarba adenosine derivatives were structurally modified to target 5-HT2B serotonin receptors as antagonists, predominantly containing branched N6-alkyl groups. N6-Dicycloalkyl-methyl groups, including their asymmetric variations, as well as 2-iodo, were found to generally favor 5-HT2Rs, while only N6-dicyclohexyl-methyl derivative 35 showed weak 5-HT2AR affinity (Ki 3.6 μM). The highest 5-HT2BR affinities were Ki 11–23 nM (N6-dicyclopropyl-methyl-2-iodo 11, 2-chloro-5′-deoxy-5′-methylthio 15 and N6-((R)-cyclobuty-cyclopropyl-methyl)-2-iodo 43), and Ki 73 nM at 5-HT2CR for 36. Direct comparison of adenine ribosides and their corresponding rigid (N)-methanocarba derivatives (cf. 51 and MRS8099 45) indicated a multifold affinity enhancement with the bicyclic ring system. Compounds 43, 45 and 48 were functional 5-HT2BR (KB 2–3 nM) and 5-HT2CR (KB 79–328 nM) antagonists in a Gq-mediated calcium flux assay, with 5-HT2BR functional selectivity ranging from 45- (48) to 113-fold (43). Substantial adenosine receptor (AR) affinity (Ki, A1AR < Ki, A3AR < Ki, A2AAR) was still present in this series, suggestive of dual acting compounds: 5-HT2B antagonist and A1AR agonist, potentially useful for treating chronic conditions (fibrosis; pain). Given its affinity (17 nM) and moderate 5-HT2BR binding selectivity (32-fold vs. 5-HT2CR, 4-fold vs. A1AR), 43 (MRS7925) could potentially be useful for anti-fibrotic therapy.

Identification of Potent, Selective, and Peripherally Restricted Serotonin Receptor 2B Antagonists from a High-Throughput Screen.

Antagonists of the serotonin receptor 2B (5-HT2B) have shown great promise as therapeutics for the treatment of pulmonary arterial hypertension, valvular heart disease, and related cardiopathies. Herein, we describe a high-throughput screen campaign that led to the identification of highly potent and selective 5-HT2B antagonists. Furthermore, selected compounds were profiled for their predicted ability to cross the blood-brain barrier. Two exemplary compounds, VU0530244 and VU0631019, were predicted to have very limited potential for brain penetration in human subjects, a critical profile for the development of 5-HT2B antagonists devoid of centrally-mediated adverse effects.

Broad Serotonergic Actions of Vortioxetine as a Promising Avenue for the Treatment of L-DOPA-Induced Dyskinesia.

Parkinson's Disease (PD) is a neurodegenerative disorder characterized by motor symptoms that result from loss of nigrostriatal dopamine (DA) cells. While L-DOPA provides symptom alleviation, its chronic use often results in the development of L-DOPA-induced dyskinesia (LID). Evidence suggests that neuroplasticity within the serotonin (5-HT) system contributes to LID onset, persistence, and severity. This has been supported by research showing 5-HT compounds targeting 5-HT1A/1B receptors and/or the 5-HT transporter (SERT) can reduce LID. Recently, vortioxetine, a multimodal 5-HT compound developed for depression, demonstrated acute anti-dyskinetic effects. However, the durability and underlying pharmacology of vortioxetine's anti-dyskinetic actions have yet to be delineated. To address these gaps, we used hemiparkinsonian rats in Experiment 1, examining the effects of sub-chronic vortioxetine on established LID and motor performance. In Experiment 2, we applied the 5-HT1A antagonist WAY-100635 or 5-HT1B antagonist SB-224289 in conjunction with L-DOPA and vortioxetine to determine the contributions of each receptor to vortioxetine's effects. The results revealed that vortioxetine consistently and dose-dependently attenuated LID while independently, 5-HT1A and 5-HT1B receptors each partially reversed vortioxetine's effects. Such findings further support the promise of pharmacological strategies, such as vortioxetine, and indicate that broad 5-HT actions may provide durable responses without significant side effects.

Role of 5-HT2 receptors family in the allergy-induced increased aorta contractile responses to 5-HT.

Asthma poses an increased risk for cardiovascular disorders, suggesting that allergy, which is an underlying process in asthma, causes atypical functioning of organs other than lungs. In a previous study in a guinea pig asthma model, we concluded that allergic sensitization increased aorta contractile responses to 5-HT. To further characterize these responses, here we explored the role of the 5-HT2 receptors family. We found that TCB-2 (5-HT2A agonist) and WAY161503 (5-HT2C agonist) induced aorta contractions resembling those elicited by 5-HT but less intense (~43 % and ~25 %, respectively). In these experiments, aortas from sensitized guinea pigs showed increased contractions to TCB-2, but not to WAY161503. In turn, MDL 100907 (5-HT2A antagonist) and RS-102221 (5-HT2C antagonist) caused a notably and a mild reduction of the 5-HT-induced contractions, respectively, with no differences seen between sensitized and non-sensitized tissues. BW723C86 (5-HT2B agonist) did not induce contractile responses and RS-127445 (5-HT2B antagonist) did not modify the contractile responses to 5-HT. In non-sensitized aortas, the pattern of protein expression of receptors was 5HT2B>5-HT2A=5-HT2C, which did not change in sensitized animals. In conclusion, we found that allergic sensitization increased the aorta contractile responses to 5-HT, partly mediated by enhanced responses of 5-HT2A receptors, which was unrelated to changes in the expression of these receptors.

Serotonin 5-HT1B receptors mediate the antidepressant- and anxiolytic-like effects of ventromedial prefrontal cortex deep brain stimulation in a mouse model of social defeat.

Deep brain stimulation (DBS) delivered to the ventromedial prefrontal cortex (vmPFC) induces antidepressant- and anxiolytic-like responses in various animal models. Electrophysiology and neurochemical studies suggest that these effects may be dependent, at least in part, on the serotonergic system. In rodents, vmPFC DBS reduces raphe cell firing and increases serotonin (5-HT) release and the expression of serotonergic receptors in different brain regions. We examined whether the behavioural responses of chronic vmPFC DBS are mediated by 5-HT1A or 5-HT1B receptors through a series of experiments. First, we delivered stimulation to mice undergoing chronic social defeat stress (CSDS), followed by a battery of behavioural tests. Second, we measured the expression of 5-HT1A and 5-HT1B receptors in different brain regions with western blot. Finally, we conducted pharmacological experiments to mitigate the behavioural effects of DBS using the 5-HT1A antagonist, WAY-100635, or the 5-HT1B antagonist, GR-127935. We found that chronic DBS delivered to stressed animals reduced the latency to feed in the novelty suppressed feeding test (NSF) and immobility in the forced swim test (FST). Though no significant changes were observed in receptor expression, 5-HT1B levels in DBS-treated animals were found to be non-significantly increased in the vmPFC, hippocampus, and nucleus accumbens and reduced in the raphe compared to non-stimulated controls. Finally, while animals given vmPFC stimulation along with WAY-100635 still presented significant responses in the NSF and FST, these were mitigated following GR-127935 administration. The antidepressant- and anxiolytic-like effects of DBS in rodents may be partially mediated by 5-HT1B receptors.

Clinical effects of Cariprazine and their relationship with polymorphisms of dopamine and serotonin receptors: preliminary results from a prospective study on schizophrenia and bipolar disorder

IntroductionCariprazine (CAR) is a D2, D3, 5HT1A receptor partial agonist and a 5HT2A, 5HT2B antagonist, used to treat Schizophrenia and Bipolar disorder. Interindividual variability in therapeutic and side effects of antipsychotics is difficult to predict, due to non-genetic and genetic factors. Single nucleotide polymorphisms (SNPs) are the main source of genetic variability, the ones in dopamine and serotonin receptors to which CAR binds are indeed likely to determine response to treatment.ObjectivesThe aim of the study is to define a relationship between CAR clinical efficacy and SNPs in dopamine and serotonin receptors genes of patients affected by schizophrenia and bipolar disorder.MethodsWe recruited 16 patients starting a monotherapy with CAR, evaluated at baseline and after 2, 4 and 8 weeks through BPRS rating scale. We selected a panel of SNPs in DR2, DR3, 5HT1A and 5HT2A receptors, with a frequency higher that 10% in Caucasians and functionally characterized. Cut-off for response to treatment was a 50% reduction of BPRS score. Statistical analysis was performed with one-way ANOVA followed by the test for linear trend between columns.ResultsAll subjects achieved response after 8 weeks of treatment, but 6 patients after 4 weeks. Early responders have a genetic profile associated with increased dopamine and serotonin receptor expression and/or binding affinity for their specific ligands. The association don’t reach statistical significance, probably due to low number of patients.ConclusionsPreliminary results suggest that an array of dopamine and serotonin receptors SNPs could predict time to respond to CAR in schizophrenia and bipolar disorder.DisclosureThe study is founded by Recordati AG, that commercialize the drug under study (Cariprazine) in Switzerland. Funding covers the costs for genetic analysis and other procedures of the study, no financial compensation is planned for investigators/authors.

Effects of the serotonin 5-HT1B receptor agonist CP 94253 on the locomotor activity and body temperature of preweanling and adult male and female rats

Serotonin 5-HT1A receptor agonists increase locomotor activity of both preweanling and adult rodents. The part played by the 5-HT1B receptor in locomotion is less certain, with preliminary evidence suggesting that the actions of 5-HT1B receptor agonists are not uniform across ontogeny. To more fully examine the role of 5-HT1B receptors, locomotor activity and axillary temperatures of preweanling and adult male and female rats was assessed. In the first experiment, adult (PD 70) and preweanling (PD 10 and PD 15) male and female rats were injected with the 5-HT1B agonist CP 94253 (2.5–10 mg/kg) immediately before locomotor activity testing and 60 min before axillary temperatures were recorded. In the second experiment, specificity of drug action was determined in PD 10 rats by administering saline, WAY 100635 (a 5-HT1A antagonist), or GR 127935 (a 5-HT1B antagonist) 30 min before CP 94253 (10 mg/kg) treatment. CP 94253 significantly increased the locomotor activity of preweanling rats on PD 10, an effect that was fully attenuated by GR 127935. Conversely, CP 94253 significantly decreased the locomotor activity of male and female adult rats, while CP 94253 did not affect the locomotor activity of PD 15 rats. Regardless of age, CP 94253 (2.5–10 mg/kg) significantly reduced the axillary temperatures of preweanling and adult rats. When considered together, these results show that 5-HT1B receptor stimulation activates motor circuits in PD 10 rats; whereas, 5-HT1B receptor agonism reduces the overall locomotor activity of adult rats, perhaps by blunting exploratory tendencies.

A Novel 5-HT1B Receptor Agonist of Herbal Compounds and One of the Therapeutic Uses for Alzheimer's Disease.

The serotonin receptor 5-HT1B is widely expressed in the central nervous system and has been considered a drug target in a variety of cognitive and psychiatric disorders. The anti-inflammatory effects of 5-HT1B agonists may present a promising approach for Alzheimer’s disease (AD) treatment. Herbal antidepressants used in the treatment of AD have shown functional overlap between the active compounds and 5-HT1B receptor stimulation. Therefore, compounds in these medicinal plants that target and stimulate 5-HT1B deserve careful study. Molecular docking, drug affinity responsive target stability, cellular thermal shift assay, fluorescence resonance energy transfer (FRET), and extracellular regulated protein kinases (ERK) 1/2 phosphorylation tests were used to identify emodin-8-O-β-d-glucopyranoside (EG), a compound from Chinese medicinal plants with cognitive deficit attenuating and antidepressant effects, as an agonist of 5-HT1B. EG selectively targeted 5-HT1B and activated the 5-HT1B-induced signaling pathway. The activated 5-HT1B pathway suppressed tumor necrosis factor (TNF)-α levels, thereby protecting neural cells against beta-amyloid (Aβ)-induced death. Moreover, the agonist activity of EG towards 5-HT1B receptor, in FRET and ERK1/2 phosphorylation, was antagonized by SB 224289, a 5-HT1B antagonist. In addition, EG relieved AD symptoms in transgenic worm models. These results suggested that 5-HT1B receptor activation by EG positively affected Aβ-related inflammatory process regulation and neural death resistance, which were reversed by antagonist SB 224289. The active compounds such as EG might act as potential therapeutic agents through targeting and stimulating 5-HT1B receptor for AD and other serotonin-related disorders. This study describes methods for identification of 5-HT1B agonists from herbal compounds and for evaluating agonists with biological functions, providing preliminary information on medicinal herbal pharmacology.

Role of peripheral 5-HT1D, 5-HT3 and 5-HT7 receptors in the mechanical allodynia induced by serotonin in mice

Serotonin (5-HT) acts as a neurotransmitter in the central nervous system (CNS) and as a mediator released by enterochromaffin cells to regulate intestinal motility. However, this amine also plays an important role as an inflammatory mediator and induces phenotypic changes of nociceptors. Despite the wide knowledge of the role of 5-HT in nociception, most studies have focused on its role in the CNS, while a clear information about its role in peripheral tissues is still lacking. In the present study, we investigated the role of peripheral 5-HT receptors in the nociceptive response induced by 5-HT or carrageenan in mice by using antagonists that target different 5-HT receptors. Mechanical nociceptive threshold was measured with an analgesimeter and evaluated after intraplantar (i.pl.) injection of 5-HT or carrageenan. 5-HT antagonists were injected via the i.pl. route. 5-HT (10, 20, 40 or 80 μg/paw) or carrageenan (100 μg/paw) induced mechanical allodynia. Pretreatment with isamoltane (5 μg; 5-HT1B antagonist) or ketanserine (1 μg; 5-HT2A antagonist) did not affect the mechanical allodynia induced by 5-HT. This response was inhibited by BRL 15572 (10 μg; 5-HT1D antagonist) or SB 269970 (25 μg; 5-HT7 antagonist). On the other hand, mechanical allodynia induced by 5-HT or carrageenan was exacerbated by ondansetron (10, 20 or 40 μg; 5-HT3 antagonist). The results indicate that activation of 5-HT1D and 5-HT7 receptors plays a role in the mechanical allodynia induced by 5-HT in mice. This study also demonstrates the inhibitory role of peripheral 5-HT3 receptors in the nociceptive response induced by 5-HT or carrageenan.

Structure activity relationship exploration of 5-hydroxy-2-(3-phenylpropyl)chromones as a unique 5-HT2B receptor antagonist scaffold

Antagonists for the serotonin receptor 2B (5-HT2B) have clinical applications towards migraine, anxiety, irritable bowl syndrome, and MDMA abuse; however, few selective 5-HT2B antagonists have been identified. Previous studies from these labs identified a natural product, 5-hydroxy-2-(2-phenylethyl)chromone (5-HPEC, 2) as the first non-nitrogenous ligand for the 5-HT2B receptor. Studies on 5-HPEC optimization led to the identification of 5-hydroxy-2-(3-phenylpropyl)chromone (5-HPPC, 3), which showed a tenfold improvement in binding affinity over 2 at 5-HT2B. This study aimed to further improve receptor pharmacology of this unique scaffold. Guided by molecular modeling studies modifications at the C-3′ and C-4′ positions of 3 were made to probe their effects on ligand binding affinity and efficacy. Among the derivatives synthesized 5-hydroxy-2-(3-(3-cyanophenyl)propyl)chromone (5-HCPC, 3d) showed the most promise with a multifold improvement in binding affinity (pKi = 7.1 ± 0.07) over 3 with retained antagonism.

Possible Involvement of Serotonergic Mechanism(s) in the Antinociceptive Effects of kaempferol

Background and Objectives: A flavonoid kaempferol (KM) exerts an anti-inflammatory effect and is reportedly capable of preventing metabolic diseases. Nonetheless, a limited number of studies have been carried out on the antinociceptive effects of kaempferol. Objectives: The present study aimed to investigate the involvement of serotonin receptors in the antinociceptive-like activity of KM in male Wistar rats using the tail-flick test. Materials and Methods: The compounds (i.e., KM, morphine, and diclofenac) were intracerebroventricularly administered to rats for the examination of central effects on the thermal pain using the tail-flick test. For the evaluation of the involvement of serotonin receptors in the possible antinociceptive effects of kaempferol, several antagonists (i.e., tropisetron, ketanserin, GR113808, WAY 100635, and penbutolol) were used. Additionally, locomotor activity and motor responses were investigated by the rotarod test after KM treatment. Results: The intracerebroventricular microinjections of KM showed antinociceptive effects using the tail-flick test. The pretreatment with tropisetron as a 5-HT3 receptor antagonist at 1 and 10 mg completely reversed the KM-related antinociception. Furthermore, ketanserin (5-HT2A receptor antagonist) and GR113808 (5-HT4 receptor antagonist) both at 10 mg reduced KM-related antinociception; however, 5-HT1A receptor antagonist WAY 100635 and 5-HT1B antagonist penbutolol did not decrease KM-related antinociception. All KM doses were not observed with a significant effect on locomotor activity or motor reactions. Conclusion: The results of the current study suggested that serotonergic receptors (i.e., 5-HT2A, 5-HT3, and 5-HT4) are effective in the KM antinociceptive activity in male rats.

Inflammation induces developmentally regulated sumatriptan inhibition of spinal synaptic transmission.

While triptans are used to treat migraine, there is evidence that they also reduce inflammation-induced pain at the spinal level. The cellular mechanisms underlying this spinal enhancement are unknown. We examined whether inflammation alters sumatriptan modulation of synaptic transmission in the rat spinal dorsal horn. Three to four days following intraplantar injection of complete Freund's adjuvant (CFA) or saline, whole cell recordings of evoked glutamatergic EPSCs were made from lumbar lamina I-II dorsal horn neurons in rat spinal slices KEY RESULTS: In 2- to 3-week-old animals, sumatriptan reduced the amplitude of evoked EPSCs and this was greater in slices from CFA, compared to saline-injected rats. In CFA-injected animals, sumatriptan increased the paired pulse ratio of evoked EPSCs and reduced the rate of spontaneous miniature EPSCs. The 5-HT1B and 5-HT1D agonists CP9 3129 and PNU109291 both inhibited evoked EPSCs in CFA but not saline-injected rats. By contrast, the 5-HT1A agonist R(+)-8-OH-DPAT inhibited evoked EPSCs in saline but not CFA-injected rats. In CFA-injected rats, the sumatriptan-induced inhibition of evoked EPSCs was reduced by the 5-HT1B and 5-HT1D antagonists NAS181 and BRL-15572. Intriguingly, the difference in sumatriptan inhibition between CFA and saline-injected animals was only observed in animals less than 4 weeks old. These findings indicate that inflammation induces a developmentally regulated 5-HT1B/1D presynaptic inhibition of excitatory transmission into the rat superficial dorsal horn. Thus, triptans could potentially act as spinal analgesic agents for inflammatory pain in the juvenile setting.

Depressive-like state sensitizes 5-HT1A and 5-HT1B auto-receptors in the dorsal raphe nucleus sub-system

Dorsal raphe (DR) and median raphe (MR) 5-HT neurons are two distinct sub-systems known to be regulated by 5-HT1A and 5-HT1B auto-receptors. Whether the auto-receptors in each sub-system are functionally altered in depressive-like state remains unknown. The present study is aimed to study a specific circuit (DR-ventral hippocampus and MR-dorsal hippocampus) within each sub-system to investigate changes in receptor sensitivity in the pathogenesis of depression. A mouse model of depression was developed through the social defeat paradigm, and was then treated with fluoxetine (FLX). 5-HT1A auto-receptor in the neuronal cell body (DR or MR) and 5-HT1B auto-receptor in the axonal terminal (ventral or dorsal hippocampus) were directly targeted by local perfusion of antagonists (5-HT1A: WAY100635; 5-HT1B: GR127935) through reverse microdialysis. Time courses of dialysate 5-HT measured at the axonal terminal were subsequently determined for each circuit. At baseline, 5-HT1A and 5-HT1B antagonists dose-dependently increased dialysate 5-HT, with sub-circuit specificity. In the depressive-like state, greater increases in dialysate 5-HT were observed only in the DR-ventral hippocampus circuit following local delivery of both antagonists, which were then fully restored following the FLX treatment. In contrast, no changes were observed in the MR-dorsal hippocampus circuit. Our results demonstrate differential changes in sensitivities of 5-HT1A and 5-HT1B auto-receptors in the DR-ventral hippocampus and MR-dorsal hippocampus circuits. 5-HT1A and 5-HT1B auto-receptors in the DR-ventral hippocampus circuit are sensitized in the depressive-like state. Taken together, these results suggest that the DR sub-system maybe the neural substrate mediating depressive phenotypes.

Adenosine A1 receptor agonist induces visceral antinociception via 5-HT1A, 5-HT2A, dopamine D1 or cannabinoid CB1 receptors, and the opioid system in the central nervous system

We have recently demonstrated that N(6)-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, acts centrally to induce a visceral antinociception. Since serotonin (5-HT), cannabinoid (CB), dopamine or opioid signaling in the central nervous system is involved in the regulation of visceral sensation, we made a hypothesis that the signaling may play a role in the CPA-induced visceral antinociception. Visceral sensation was evaluated by colonic distension-induced abdominal withdrawal reflex (AWR) in conscious rats. Subcutaneously administered CPA significantly increased the threshold of colonic distension-induced AWR. Intracisternal injection of either 5-HT1A or 5-HT2A receptor antagonist blocked the CPA-induced visceral antinociception while 5-HT1B antagonist did not block the CPA-induced visceral antinociception. Subcutaneous injection of dopamine D1 receptor antagonist, CB1 receptor antagonist or naloxone significantly blocked the CPA-induced visceral antinociception while neither subcutaneous injection of dopamine D2 receptor antagonist nor CB2 receptor antagonist blocked the CPA-induced anti-pain action. These results suggest that 5-HT1A, 5-HT2A, dopamine D1, CB1 receptors and the opioid system in the CNS may specifically mediate the CPA-induced visceral antinociception. These findings may help in understanding the physiological relevance of central adenosine with special reference to the pathophysiology of altered visceral sensation especially in irritable bowel syndrome.

Functional characterization of 5-HT1A and 5-HT1B serotonin receptor signaling through G-protein-activated inwardly rectifying K+ channels in a fluorescence-based membrane potential assay

The 5-HT1A and 5-HT1B serotonin receptors are abundantly expressed in the CNS and constitute validated as well as putative drug targets in a variety of psychiatric and cognitive disorders, alcoholism/addiction, pain and migraine. In the present study we have characterized the functional properties of human 5-HT1A and 5-HT1B stably co-expressed with the human G-protein-activated inwardly rectifying K+ channel 2 (GIRK2) in HEK293 cells in the fluorescence-based FLIPR® Membrane Potential Blue (FMP) assay. Serotonin and other agonists induced robust decreases in fluorescence levels in the 5-HT1A/GIRK2- and 5-HT1B/GIRK2-HEK293 cells in a concentration-dependent manner in the assay, and these responses could be inhibited by selective 5-HT1A/5-HT1B antagonists and by the Gαi/o-protein inhibitor pertussis toxin (PTX). Five additional stable HEK293 cell lines co-expressing 5-HT1A or 5-HT1B with GIRK2 and one of the PTX-insensitive Gαi/o-subunit mutants Gαi1C351I, Gαi2C352I and Gαo1C351I were constructed, and 5-HT1A/5-HT1B-mediated responses through these specific Gαi/o-subunits were measured in these cells pretreated with PTX in the FMP assay. The functional properties of 16 reference 5-HT1 agonists were characterized at the seven cell lines, which constitutes the most detailed pharmacological profiling and comparison of 5-HT1A and 5-HT1B receptor signaling in the same assay published to date. We propose that this approach to assay 5-HT1-mediated signaling through endogenous Gαi/o-proteins in HEK293 cells or through specific Gαi/o-subunits in a fairly high-throughput manner holds some advantages to other functional assays for Gαi/o-coupled receptors. The assay will facilitate detailed profiling of the Gαi/o- and Gβγ-mediated signaling of 5-HT1A and 5-HT1B at the molecular level, and it could also be used to identify novel modulators for the receptors.

P.106 Induction of aggregation by tau seeding in tauP301S-PS19 transgenic mice

Acute ethanol (EtOH) consumption exerts a biphasic effect on behavior and increases serotonin levels in the brain. However, the molecular mechanisms underlying alcohol-mediated behavioral responses still remain to be fully elucidated. Here, we investigate pharmacologically the involvement of the serotonergic pathway on acute EtOH-induced behavioral changes in zebrafish. We exposed zebrafish to 0.25, 0.5, 1.0% (v/v) EtOH for 1 h and analyzed the effects on aggression, anxiety-like behaviors, and locomotion. EtOH concentrations that changed behavioral responses were selected to the subsequent experiments. As a pharmacological approach, we used pCPA (inhibitor of tryptophan hydroxylase), WAY100135 (5-HT1A antagonist), buspirone (5-HT1A agonist), CGS12066A and CGS12066B (5-HT1B antagonist and agonist, respectively), ketanserin (5-HT2A antagonist) and (±)-DOI hydrochloride (5-HT2A agonist). All serotonergic receptors tested modulated aggression, with a key role of 5-HT2A in aggressive behavior following 0.25% EtOH exposure. Because CGS12066B mimicked 0.5% EtOH anxiolysis, which was antagonized by CGS12066A, we hypothesized that anxiolytic-like responses are possibly mediated by 5-HT1B receptors. Conversely, the depressant effects of EtOH are probably not related with direct changes on serotonergic pathway. Overall, our novel findings demonstrate a role of the serotonergic system in modulating the behavioral effects of EtOH in zebrafish. These data also reinforce the growing utility of zebrafish models in alcohol research and help elucidate the neurobiological mechanisms underlying alcohol abuse and associated complex behavioral phenotypes.

Serotonin 5-HT1A and 5-HT1B receptors co-mediate the RU 24969-induced locomotor activity of male and female preweanling rats

The serotonin (5-HT) 1A/1B agonist RU 24969 robustly increases the locomotor activity of adult male rats and mice; however, studies using selective antagonists alternately report that 5-HT1A, 5-HT1B, or both receptor types mediate RU 24969's locomotor activating effects. The purpose of the present study was to extend these past findings by administering a selective 5-HT1 agonist and/or antagonists to male and female preweanling rats. This age group was tested because younger rats often exhibit psychopharmacological responses that are quantitatively or qualitatively different from adult rats. In a series of experiments, male and female preweanling rats were pretreated with vehicle, the 5-HT1A antagonist WAY 100635 (0.5, 1, 5, or 10 mg/kg), or the 5-HT1B antagonists NAS-181 (5 or 10 mg/kg) or SB 216641 (5 or 10 mg/kg) 30 min before assessment of locomotor activity. Rats were injected with saline or RU 24969 immediately prior to testing. Results showed that RU 24969 (0.625, 1.25, 2.5, or 5 mg/kg) significantly increased the locomotor activity of both male and female preweanling rats (no sex differences were apparent). Antagonism of either the 5-HT1A or the 5-HT1B receptor was sufficient to significantly reduce the locomotor activity of RU 24969-treated preweanling rats. Unexpectedly, NAS-181 did not act as a silent receptor antagonist, as both doses of NAS-181 significantly increased the locomotor activity of saline-treated preweanling rats. In sum, the present results show that both the 5-HT1A and 5-HT1B receptor systems mediate locomotion during the late preweanling period, and this mediation does not vary according to sex.

Role of the serotonergic system in ethanol-induced aggression and anxiety: A pharmacological approach using the zebrafish model

Acute ethanol (EtOH) consumption exerts a biphasic effect on behavior and increases serotonin levels in the brain. However, the molecular mechanisms underlying alcohol-mediated behavioral responses still remain to be fully elucidated. Here, we investigate pharmacologically the involvement of the serotonergic pathway on acute EtOH-induced behavioral changes in zebrafish. We exposed zebrafish to 0.25, 0.5, 1.0% (v/v) EtOH for 1 h and analyzed the effects on aggression, anxiety-like behaviors, and locomotion. EtOH concentrations that changed behavioral responses were selected to the subsequent experiments. As a pharmacological approach, we used pCPA (inhibitor of tryptophan hydroxylase), WAY100135 (5-HT1A antagonist), buspirone (5-HT1A agonist), CGS12066A and CGS12066B (5-HT1B antagonist and agonist, respectively), ketanserin (5-HT2A antagonist) and (±)-DOI hydrochloride (5-HT2A agonist). All serotonergic receptors tested modulated aggression, with a key role of 5-HT2A in aggressive behavior following 0.25% EtOH exposure. Because CGS12066B mimicked 0.5% EtOH anxiolysis, which was antagonized by CGS12066A, we hypothesized that anxiolytic-like responses are possibly mediated by 5-HT1B receptors. Conversely, the depressant effects of EtOH are probably not related with direct changes on serotonergic pathway. Overall, our novel findings demonstrate a role of the serotonergic system in modulating the behavioral effects of EtOH in zebrafish. These data also reinforce the growing utility of zebrafish models in alcohol research and help elucidate the neurobiological mechanisms underlying alcohol abuse and associated complex behavioral phenotypes.

The convergence of fibrosis and immune pathways: 5-HT2 and 5-HT2B antagonists attenuate profibrotic phenotype in human pancreatic stellate cells by modulating signal transducer and activator of transcription 3 phosphorylation

Background: Immune mechanisms are believed to play an important role in the pathogenesis of pancreatic inflammation and fibrosis. Transforming growth factor β1-mediated mechanisms are well known; however, an effective treatment option is yet not available. Serotonin (5-HT) is known to induce fibrosis in carcinoid tumors and diseases such as scleroderma and blocking 5-HT has shown promise in the attenuation of fibrosis in animal models of scleroderma. Herein, we evaluated the role of 5-HT in pancreatic fibrosis and its reversal with antagonists of 5-HT. Methodology: Pancreatic stellate cells (PSCs) were cultured from the two patients of pancreatic cancer undergoing pancreaticoduodenectomy. PSCs were cultured in the presence of 5-HT and as per the disease mimicking strategy (PSCs cultured with 5-HT first followed by 5-HT antagonist (terguride) and 5-HT2Bantagonist (SB204741) for 24 h and pretreatment strategy, PSCs treated with 5-HT antagonists first followed by 5-HT. mRNA expression of profibrotic genes (col1A1, Col1A2, ACTA-2, and CTGF) and Western blot analysis of signal transducer and activator of transcription 3 (STAT-3) and Smad-3 proteins was carried out. Results: 5-HT-induced profibrotic genes mRNA expression in PSCs was significantly reversed by 5-HT antagonists, terguride and SB204741, to a large extent. Pretreatment strategy was more effective in the amelioration of profibrotic potential. SB204741, 5-HT2Bantagonist was more effective than terguride. 5-HT antagonists mediated their effect by reducing the phosphorylation of noncanonical pathway mediator pSTAT3 more significantly then canonical pathway mediator pSmad-3. Conclusion: 5-HT induces profibrotic potential in human PSCs and inhibitors of 5-HT receptors, Terguride and SB 204,741 ameliorate it by reducing the phosphorylation of STAT-3 and Smad-3, more so in pretreatment strategy. 5-HT inhibitors may have a potential to ameliorate fibrosis in pancreas.