Search

Ova disertacija napravljena je kako bi se istražio utjecaj različitih podražaja, ali i uloge 5-HT i ɑ2-adrenergičnih receptora u razvoju respiracijske plastičnosti. Istraživanje je podijeljeno u dva dijela. U prvom dijelu, uspoređeni su dugoročni učinci ponavljanih i kontinuiranih hiperkapnijskih i hipoksijskih podražaja te ponavljanog kombiniranog hiperkapnijsko-hipoksijskog podražaja na plastičnost freničnog živca. U drugom dijelu, različiti agonisti i antagonisti 5-HT i ɑ2 recceptora su mikroinjicirani u kaudalno rafe područje kako bi se ispitala uloga receptora važnih u nastanku i održavanju pLTD-a. Odrasli, muški, uretanom anestezirani, vagotomizirani, paralizirani, mehanički ventilirani Sprague-Dawley štakori bili su izloženi: akutnoj ponavljanoj hiperkapniji (AIHczrak ili AIHcO2), akutnoj ponavljanoj hipoksiji (AIH), kombiniranoj ponavljanoj hiperkapniji i hipoksiji (AIHcH), kontinuiranoj hiperkapniji (CHc), ili kontinuiranoj hipoksiji (CH). U drugom dijelu studije štakori su bili izloženi protokolu akutne ponavljane hiperkapnije (5 epizoda 15% CO2 u zraku, svaka epizoda trajala je 3 minute). Eksperimentalnoj skupini mikroinjiciran je ili selektivni agonist 5-HT1A receptora 8-OH-DPAT ili nespecfični antagonist 5-HT receptora metizergid ili α2-adrenergični antagonisti johimbin, dok je kontrolnoj skupini mikroinjicirana fiziološka otopina u kaudalno rafe područje. U AIHc skupini, frekvencija freničnog živca smanjena je s 44.25 ± 4.06 u T0 na 35.29 ± 5.21 udisaja / min u T60, (P = 0.04, AIHczrak) i sa 45.5 ± 2.62 na 37.17 ± 3.68 udisaja/min (P=0.05, AIHcO2). tj. izazvana je dugoročna depresija frekvencije freničkog živca. Izloženost AIH izazvala je povećanje pPNA u T60 za 143.7 ± 30.1% u usporedbi s početnim vrijednostima, P=0.02), tj. izazvana je dugoročna facilitacija freničkog živca. Kombinirana ponavljana hiperkapnija i hipoksija ili kontinuirana hiperkapnija ili hipoksija nisu izazvale dugoročnu plastičnost freničnog živca. U drugom dijelu istraživanja, u kontrolnoj skupini, pPNA se smanjila 60 minuta nakon završetka posljednjeg hiperkapnijskog podražaja u usporedbi s osnovnim vrijednostima, tj. razvio se pLTD (P=0.02). U 8-OH-DPAT skupini, pPNA je značajno smanjen u T15, T30 i T60 u usporedbi s početnim vrijednostima, tj. nastao je pLTD (P=0.01). U skupini koja je primala metizergid, akutna ponavljana hiperkapnija nije izazvala značajne promjene u pPNA u T15, T30 i T60 u usporedbi s osnovnim vrijednostima. U skupini s johimbinom, akutna ponavljana hiperkapnija nije izazvala značajne promjene vršne aktivnosti freničkog živca u T15, T30 i T60 u usporedbi s osnovnim vrijednostima nakon kraja posljednje hiperkapnijske epizode. Zaključno, ponavljani hiperkapnijski ili hipoksijski podražaji potrebni su za izazivanje plastičnosti freničnog živca. Aktivacija 5-HT1A receptora važna je za nastanak pLTD, dok blokiranje α2-adrenergičkih receptora sprječava razvoj pLTD-a nakon akutne ponavljane hiperkapnije. Ovi rezultati sugeriraju da kemijska modulacija 5-HT i α2-adrenergičkih receptora u rafe jezgri utječe na hiperkapnijom izazvan pLTD, nudeći važan uvid u razumijevanje mehanizama uključenih u razvoj respiratorne plastičnosti., This study was undertaken to determine pattern sensitivity of phrenic nerve plasticity in respect to different respiratory challenges and to investigate the role of 5-HT and α2-adrenergic receptors in the initiation of pLTD. Study was divided into two parts. In the first, we compared long-term effects of intermittent and continuous hypercapnic and hypoxic stimuli, and combined intermittent hypoxia and hypercapnia on phrenic nerve plasticity. In the second part, different drugs were microinjected in the raphe region to investigate which receptors are important for pLTD induction and maintance. Adult, male, urethane-anesthetized, vagotomized, paralyzed, mechanically ventilated Sprague-Dawley rats were exposed to: acute intermittent hypercapnia (AIHcair or AIHcO2), acute intermittent hypoxia (AIH), combined intermittent hypercapnia and hypoxia (AIHcH), continuous hypercapnia (CHc), or continuous hypoxia (CH). In the second part of the study rats were exposed to the protocol of acute intermittent hypercapnia (5 episodes of 15% CO2 in air, each episode lasting 3 minutes). Experimental group received microinjection of selective 5-HT1A receptors agonist 8-OH-DPAT, broad spectrum 5-HT antagonist methysergide or α2- adrenergic antagonist yohimbine, whereas control group received microinjection of 0.9% saline into the caudal raphe region. Peak phrenic nerve activity (pPNA) and burst frequency were analyzed during baseline (T0), hypercapnia or hypoxia exposures, at 15, 30, and 60 minutes (T60) after the end of the stimulus. In the AIHc groups phrenic nerve frequency decreased from 44.25 ± 4.06 at T0 to 35.29 ± 5.21 breaths/min at T60, (P=0.04, AIHcair) and from 45.5 ± 2.62 to 37.17 ± 3.68 breaths/min (P=0.05, AIHcO2), i.e. frequency phrenic long term depression was induced. Exposure to AIH elicited increase of pPNA at T60 by 143.7 ± 30.1 % compared to baseline (P=0.02), i.e. phrenic long-term facilitation was induced. Combined intermittent hypercapnia and hypoxia or continuous hypercapnia or hypoxia failed to induce long-term plasticity of the phrenic nerve. In the second part of the study, in the control group, pPNA decreased 60 minutes after the end of the last hypercapnic episode compared to baseline values, i.e. pLTD developed (P=0.02). In the 8-OH-DPAT group pPNA significantly decreased at T15, T30 and T60 compared to baseline, i.e. pLTD developed (P=0.01). In the methysergide-treated group, acute intermittent hypercapnia did not evoke significant changes in pPNA at T15, T30, and T60 compared to baseline values. In the yohimbine group acute intermittent hypercapnia did not evoke significant changes of the peak phrenic nerve activity at T15, T30, and T60 compared to baseline values after the end of the last hypercapnic episode. In conclusion, intermittency of the hypercapnic or hypoxic stimuli is needed to evoke phrenic nerve plasticity. Activation of 5-HT1A receptors accentuated induction of pLTD, whereas blockade of α2-adrenergic receptors prevented development of pLTD following acute intermittent hypercapnia in anesthetized rats. These results suggest that chemical modulation of 5-HT and α2-adrenergic receptors in raphe nuclei affects hypercapnia induced pLTD, offering the important insights in understanding the mechanisms involved in development of respiratory plasticity.

Depression is one of the most common and widespread medical issues and major depression is also one of the most disabling of all medical conditions. Although enormous efforts have been invested in research, there is no evidence that an abnormality in serotonin levels or other monoamines causes depression or that certain people are genetically predisposed to produce too little serotonin and therefore to experience depression. As Allen Frances, the Chair of the Diagnostic and Statistical Manual IV (DSM-IV) Task Force said: “Billions of research dollars have failed to produce convincing evidence that any mental disorder is a discrete disease entity with a unitary cause. Dozens of different candidate genes have been “found,” but in follow-up studies, each turned out to be fool’s gold.” Now we just have to figure out WHAT we can trust. Proper education for the students and teachers is one approach.

We analyzed the expression of the serotonin receptors 5-HT1A, 5-HT2A, and 5-HT3A at four different stages of fetal lung development from 12 to 40 weeks of gestation, divided into four groups: the pseudoglandular stage (12–16th week of development; n = 8), the canalicular stage (16th–26th week of development; n = 7), the saccular stage (26th-36th week of development; n = 5), and the alveolar stage (36th–40th week of development; n = 5). The strongest expression of all three receptor types was found in the epithelium of the proximal airways during the pseudoglandular, canalicular, and saccular stages and in a vascular wall. 5-HT1A was also strongly expressed in the smooth muscle cells of the proximal airway. Vascular smooth muscle cells and endothelium occasionally showed a strong expression of 5-HT1A and 5-HT2A. In the alveolar stage, the expression of 5-HT1A, 5-HT2A, and 5-HT3A was detected in both type I (p1) and type II (p2) pneumocytes, with a stronger expression in p2. A significant decrease in percent the 5-HT2A area and in the integrated density was observed at the alveolar stage. On the other hand, a significant decrease in the percentage area but an increase in the integrated density was observed for 5-HT3A toward the alveolar stage, suggesting that a smaller number of cells expressed 5-HT3A but that they (p1 and p2) significantly increased their 5-HT3A expression at the alveolar stage. The results presented provided us with new data on the development and function of the serotonin system in the human fetal lung and gave us insight into their possible involvement in the pathogenesis of lung pathology, particularly that characteristic of the neonatal period.

A series of 15 new derivatives of 6-acetyl-7-hydroxy-4-methylcoumarin containing a piperazine group were designed with the help of computational methods and were synthesized to study their affinity for the serotonin 5-HT1A and 5-HT2A receptors. Among them, 6-acetyl-7-{4-[4-(3-bromophenyl)piperazin-1-yl]butoxy}-4-methylchromen-2-one (4) and 6-acetyl-7-{4-[4-(2-chlorophenyl)piperazin-1-yl]butoxy}-4-methylchromen-2-one (7) exhibited excellent activity for 5-HT1A receptors with Ki values 0.78 (0.4–1.4) nM and 0.57 (0.2–1.3) nM, respectively, comparable to the Ki values of 8-OH-DPAT (0.25 (0.097–0.66) nM). The equilibrium dissociation constant values of the tested compounds showed differential intrinsic activities of the agonist and antagonist modes.

Antipsychotic-induced parkinsonism (AIP) is the most common type of extrapyramidal side effect (EPS), caused by the blockage of dopamine receptors. Since dopamine availability might influence the AIP risk, the dopamine transporter (DAT) and serotonin receptors (5-HTRs), which modulate the dopamine release, may be also involved in the AIP development. As some of the individual differences in the susceptibility to AIP might be due to the genetic background, this study aimed to examine the associations of SLC6A3, HTR2C and HTR6 gene polymorphisms with AIP in haloperidol-treated schizophrenia patients. The Extrapyramidal Symptom Rating Scale (ESRS) was used to evaluate AIP as a separate entity. Genotyping was performed using a PCR, following the extraction of blood DNA. The results revealed significant associations between HTR6 rs1805054 polymorphism and haloperidol-induced tremor and rigidity. Additionally, the findings indicated a combined effect of HTR6 T and SLC6A3 9R alleles on AIP, with their combination associated with significantly lower scores of ESRS subscale II for parkinsonism, ESRS-based tremor or hyperkinesia and ESRS subscales VI and VIII. These genetic predictors of AIP could be helpful in better understanding its pathophysiology, recognizing the individuals at risk of developing AIP and offering personalized therapeutic strategies for the patients suffering from this EPS.

The biogenic monoamine serotonin (5-hydroxytryptamine, 5-HT) is a chemical messenger widely distributed in the brain and various other organs. Its homeostasis is maintained by the coordinated activity of a variety of proteins, including enzymes of serotonin metabolism, transmembrane transporters of serotonin, and serotonin receptors. The serotonin system has been identified also in the placenta in rodent models as a key component of placental physiology. However, serotonin pathways in the human placenta are far from well understood. Their alterations may have long-lasting consequences for the fetus that can manifest later in life. In this review, we summarize information on the location of the components of the serotonin system in the human placenta, their regulation, function, and alterations in pathological pregnancies. We highlight current controversies and discuss important topics for future research.

There is increasing interest in the therapeutic potential of psilocybin. In rodents, the serotonin precursor, 5-hydroxytryptophan (5-HTP) and psilocybin induce a characteristic 5-HT2A receptor (5-HT2AR)-mediated head twitch response (HTR), which is correlated with the human psychedelic trip. We examined the role of other serotonergic receptors and the trace amine -associated receptor 1 (TAAR1) in modulating 5-HTP- and psilocybin-induced HTR. Male C57BL/6J mice (11 weeks, ~30 g) were administered 5-HTP, 50–250 mg/kg i.p., 200 mg/kg i.p. after pretreatment with 5-HT/TAAR1 receptor modulators, psilocybin 0.1–25.6 mg/kg i.p. or 4.4 mg/kg i.p., immediately preceded by 5-HT/TAAR1 receptor modulators. HTR was assessed in a custom-built magnetometer. 5-HTP and psilocybin induced a dose-dependent increase in the frequency of HTR over 20 min with attenuation by the 5-HT2AR antagonist, M100907, and the 5-HT1AR agonist, 8-OH-DPAT. The 5-HT2CR antagonist, RS-102221, enhanced HTR at lower doses but reduced it at higher doses. The TAAR1 antagonist, EPPTB, reduced 5-HTP- but not psilocybin-induced HTR. We have confirmed the key role of 5-HT2AR in HTR, an inhibitory effect of 5-HT1AR, a bimodal contribution of 5-HT2CR and a role of TAAR1 in modulating HTR induced by 5-HTP. Compounds that modulate psychedelic-induced HTR have important potential in the emerging therapeutic use of these compounds.

Background: Hypoxic–ischemic (HI) insults have important deleterious consequences in newborns, including short-term morbidity with neuromotor and cognitive disturbances. Cannabidiol (CBD) has demonstrated robust neuroprotective effects and shows anxiolytic/ antidepressant effects as well. These effects are thought to be related to serotonin 5-HT1A receptor (5HT1AR) activation. We hereby aimed to study the role of 5HT1AR in the neuroprotective and behavioral effects of CBD in HI newborn piglets. Methods: 1-day-old piglets submitted to 30 min of hypoxia (FiO2 10%) and bilateral carotid occlusion were then treated daily with vehicle, CBD 1 mg/kg, or CBD with the 5HT1AR antagonist WAY 100635 1 mg/kg 72 h post-HI piglets were studied using amplitude-integrated EEG to detect seizures and a neurobehavioral test to detect neuromotor impairments. In addition, behavioral performance including social interaction, playful activity, hyperlocomotion, and motionless periods was assessed. Then, brain damage was assessed using histology (Nissl and TUNEL staining) and biochemistry (proton magnetic resonance spectroscopy studies. Results: HI led to brain damage as assessed by histologic and biochemistry studies, associated with neuromotor impairment and increased seizures. These effects were not observed in HI piglets treated with CBD. These beneficial effects of CBD were not reversed by the 5HT1AR antagonist, which is in contrast with previous studies demonstrating that 5HT1AR antagonists eliminated CBD neuroprotection as assessed 6 h after HI in piglets. HI led to mood disturbances, with decreased social interaction and playfulness and increased hyperlocomotion. Mood disturbances were not observed in piglets treated with CBD, but in this case, coadministration of the 5HT1AR antagonist eliminates the beneficial effects of CBD. Conclusion: CBD prevented HI-induced mood disturbances in newborn piglets by acting on 5HT1AR. However, 5HT1AR activation seems to be necessary for CBD neuroprotection only in the first hours after HI., PI19/00927 project ISCIII European Regional Development Fund (ERDF) “A way to make Europe

Memory impairments constitute a significant problem worldwide, and the COVID-19 pandemic dramatically increased the prevalence of cognitive deficits. Patients with cognitive deficits, specifically memory disturbances, have underlying comorbid conditions such as schizophrenia, anxiety, or depression. Moreover, the available treatment options have unsatisfactory effectiveness. Therefore, there is a need to search for novel procognitive and anti-amnesic drugs with additional pharmacological activity. One of the important therapeutic targets involved in the modulation of learning and memory processes are serotonin receptors, including 5-HT1A, 5-HT6, and 5-HT7, which also play a role in the pathophysiology of depression. Therefore, this study aimed to assess the anti-amnesic and antidepressant-like potential of JJGW08, a novel arylpiperazine alkyl derivative of salicylamide with strong antagonistic properties at 5-HT1A and D2 receptors and weak at 5-HT2A and 5-HT7 receptors in rodents. First, we investigated the compound’s affinity for 5-HT6 receptors using the radioligand assays. Next, we assessed the influence of the compound on long-term emotional and recognition memory. Further, we evaluated whether the compound could protect against MK-801-induced cognitive impairments. Finally, we determined the potential antidepressant-like activity of the tested compound. We found that JJGW08 possessed no affinity for 5-HT6 receptors. Furthermore, JJGW08 protected mice against MK-801-induced recognition and emotional memory deficits but showed no antidepressant-like effects in rodents. Therefore, our preliminary study may suggest that blocking serotonin receptors, especially 5-HT1A and 5-HT7, might be beneficial in treating cognitive impairments, but it requires further investigation.

Serotonergic receptors are affected in Alzheimer's disease (AD). The overall decrease in the serotonergic transmission is also observed in AD. Previous studies showed the association of polymorphisms in genes for serotonin (5HT) receptors with behavioral and psychological symptoms of dementia (BPSD). In this study, we aimed to test if individuals carrying different genotypes in 5HTR2A rs 6313 (T102C), 5HTR1B rs13212041, 5HTR6 rs1805054 (C267T), and 5HTR2C rs 3813929 (-795C/T) polymorphisms have a higher risk for development of AD. Cerebrospinal fluid (CSF) AD biomarkers, neuropsychological tests, and apolipoprotein E (APOE) haplotype were determined in 115 AD patients and 53 mildly cognitively impaired patients, while 2701 cognitively healthy individuals with determined APOE haplotype were also included. We observed that all four analyzed polymorphisms in genes for 5HT receptors showed an association with either CSF, neuropsychological or genetic biomarkers of AD. Thus, the potential of these polymorphisms as early genetic biomarkers of AD should be further investigated.

Serotonin is a key neurotransmitter that is implicated in a wide variety of behavioral and cognitive phenotypes. Originating in the raphe nuclei, 5-HT neurons project widely to innervate many brain regions implicated in the functions. During the development of the brain, as serotonin axons project and innervate brain regions, there is evidence that 5-HT plays key roles in wiring the developing brain, both by modulating 5-HT innervation and by influencing synaptic organization within corticolimbic structures. These actions are mediated by 14 different 5-HT receptors, with region- and cell-specific patterns of expression. More recently, the role of the 5-HT system in synaptic re-organization during adulthood has been suggested. The 5-HT neurons have the unusual capacity to regrow and reinnervate brain regions following insults such as brain injury, chronic stress, or altered development that result in disconnection of the 5-HT system and often cause depression, anxiety, and cognitive impairment. Chronic treatment with antidepressants that amplify 5-HT action, such as selective serotonin reuptake inhibitors (SSRIs), appears to accelerate the rewiring of the 5-HT system by mechanisms that may be critical to the behavioral and cognitive improvements induced in these models. In this review, we survey the possible 5-HT receptor mechanisms that could mediate 5-HT rewiring and assess the evidence that 5-HT-mediated brain rewiring is impacting recovery from mental illness. By amplifying 5-HT-induced rewiring processes using SSRIs and selective 5-HT agonists, more rapid and effective treatments for injury-induced mental illness or cognitive impairment may be achieved.

Tese de Doutoramento em Ciências da Saúde, A doença de Machado-Joseph (DMJ) é uma doença debilitante caracterizada pela perda progressiva do equilíbrio e da coordenação motora. A nível molecular, a DMJ está associada à expansão da repetição de Citosina-Adenina-Guanina (CAG) no gene da ataxina-3. Isto traduz-se num segmento expandido de poliglutaminas na proteína ataxina-3 (ATXN3), que é propenso à auto-associação, sendo a agregação desta proteína uma característica da doença. Apesar dos vários esforços efectuados, não existe cura para esta doença fatal. A modulação da sinalização serotoninérgica foi anteriormente descrita como potencial estratégia terapêutica para a DMJ. O tratamento com citalopram (CIT), um inibidor seletivo da recaptação de serotonina, suprimiu a patogénese da DMJ em vários modelos animais, sendo o efeito do CIT dependente do transportador de serotonina, o seu alvo molecular, com provável contribuição dos receptores serotoninérgicos 5-HT1A/SER-4 e 5-HT2/SER-1. No entanto, o papel de cada receptor serotoninérgico na supressão da proteotoxicidade da ATXN3 mutante não foi ainda elucidado. Aqui, com o objetivo de determinar a contribuição de cada receptor de serotonina (5-HTR) na supressão da proteotoxicidade da ATXN3 usámos um modelo de C. elegans que recapitula as principais características da DMJ, seguindo abordagens químico-genéticas, farmacológicas e bioquímicas, bem como técnicas de microscopia in vivo. Inicialmente, explorámos a contribuição do receptor 5-HT1A usando befiradol, um agonista potente e altamente específico destes receptores. A administração crónica e aguda deste composto melhorou a função motora dos mutantes, sendo o ortólogo do receptor 5-HT1A no nematode, o SER-4, necessário para a sua ação. De forma a aprofundar o conhecimento sobre o impacto do tratamento com befiradol na proteotoxicidade da ATXN3, optimizámos ensaios de retardação em filtro e fracionamento bioquímico para avaliar as espécies agregadas da ATXN3, demonstrando-se que ambas as modalidades de tratamento afectaram a solubilidade desta proteína. De realçar que, usando químicogenética e medições de atividade neuronal, foi-nos possível definir os auto- e hetero-receptores 5- HT1A/SER-4 como essenciais para o efeito terapêutico do befiradol e propor o receptor 5-HT1A como novo alvo terapêutico para a DMJ. Além disso, identificámos uma contribuição parcial dos receptores 5- HT6/SER-5 e 5-HT7/SER-7 para a ação do CIT. Surpreendentemente, o antagonismo dos receptores 5- HT2/SER-1, 5-HT6/SER-5 e 5-HT7/SER-7 mostrou-se benéfico para a função motora de animais mutantes, o que é consistente com dados de perfil de expressão de RNA e farmacoterapia multimodal obtidos no nosso laboratório. Embora promissores, são necessários mais estudos para reforçar estes achados e elucidar as vias específicas pelas quais cada 5-HTR individual afecta a proteotoxicidade da ATXN3., Machado-Joseph disease (MJD) is a debilitating disease mainly characterized by a progressive loss of balance and motor coordination. At the molecular level, this autosomal dominant disease is associated with an expansion of the Cytosine-Adenine-Guanine (CAG) repeat in the ataxin-3 gene. This translates into a pathologic polyglutamine tract in the ataxin-3 protein (ATXN3) that is prone to selfassociate, aggregation of ATXN3 being a hallmark of the disease. Although several efforts have been made, no cure is yet available for this fatal disorder. Serotonergic signalling modulation, by selective serotonin reuptake inhibitors (SSRIs), was previously described as a potential therapeutical strategy for MJD. Treatment with citalopram (CIT), a SSRI used in the clinics to treat depression, suppressed MJD pathogenesis in a disease-modifying manner, in several animal models. CIT’s effect was dependent on its molecular target, the serotonin transporter SERT, likely with the additional need of the serotonin receptors 5-HT1AR/SER-4 and 2-HT2R/SER-1. However, the exact role of each serotonin receptor (5-HTR) in the suppression of mutant ATXN3 proteotoxicity remains to be fully elucidated. Here, we aimed at determining the contribution of each of the 5-HTRs in the suppression of ATXN3 proteotoxicity using a C. elegans model that recapitulates major hallmarks of MJD, by employing chemical genetics, pharmacological- and biochemical- approaches, as well as in vivo dynamic imaging techniques. First, we explored the contribution of the 5-HT1AR using a highly specific and potent agonist of these receptors, befiradol. Chronic and acute administration of befiradol rescued the motor function of mutant animals, the 5-HT1AR orthologue in the nematode, SER-4, being required for its action. To further explore the impact of befiradol treatment in ATXN3 proteotoxicity, we optimised filter retardation and biochemical fractionation assays to assess mutant ATXN3 aggregation states, and we showed that both treatment modalities impacted ATXN3 solubility. Importantly, using chemical genetics and single neuron neuronal activity measurements, we defined 5-HT1AR/SER-4 auto- and heteroreceptors’ function to be essential to befiradol therapeutic outcome, and the 5-HT1AR as a novel therapeutic target for MJD. Additionally, we found a partial contribution of 5-HT6R/SER-5 and 5-HT7R/SER-7 for CIT action and, surprisingly, antagonism of 5-HT2R/SER-1, 5-HT6R/SER-5 and 5-HT7R/SER-7 showed to be beneficial for motor function of mutant animals, which is consistent with RNA-expression profiling and multimodal pharmacotherapy results obtained in our laboratory. Although promising, further studies are needed to support these findings and to elucidate the specific pathways by which each individual serotonin receptor impacts ATXN3 proteotoxicity., The work presented in this thesis was performed at the Life and Health Sciences Research Institute (ICVS) and at the Bn’ML – Behavioral & Molecular Lab, at the School of Medicine, University of Minho. Financial support was provided by grants from the Portuguese Foundation for Science and Technology (FCT): Doctoral Programme in Applied Health Sciences fellowship (PD/BDE/127834/2016) to J. Pereira-Sousa; and by FEDER, through the Competitiveness Internationalization Operational Programme (POCI) under the scope of the project POCI-01-0145-FEDER-031987 to A. Teixeira-Castro, supported by North Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER022122); and by National funds, through the FCT - project UIDB/50026/2020 and UIDP/50026/2020. This work was also funded through the National Ataxia Foundation (NAF), USA.

Background: The current study utilizes in silico molecular docking/molecular dynamics to evaluate the binding affinity of apigenin and safranal with 5HT1AR/5HT2AR, followed by assessment of in vivo effects of these compounds on depressive and anxious behavior. Methods: The docking between apigenin and safranal and the 5HT1A and 5HT2A receptors was performed utilizing AutoDock Vina software, while MD and protein-lipid molecular dynamics simulations were executed by AMBER16 software. For in vivo analysis, healthy control (HC), disease control (DC), fluoxetine-, and apigenin-safranal-treated rats were tested for changes in depression and anxiety using the forced swim test (FST) and the elevated plus-maze test (EPMT), respectively. Results: The binding affinity estimations identified the superior interacting capacity of apigenin over safranal for 5HT1A/5HT2A receptors over 200 ns MD simulations. Both compounds exhibit oral bioavailability and absorbance. In the rodent model, there was a significant increase in the overall mobility time in the FST, while in the EPMT, there was a decrease in latency and an increase in the number of entries for the treated and HC rats compared with the DC rats, suggesting a reduction in depressive/anxiety symptoms after treatment. Conclusions: Our analyses suggest apigenin and safranal as prospective medication options to treat depression and anxiety.

Several studies confirmed the reciprocal interactions between adrenergic and serotoninergic systems and the influence of these phenomena on the pathogenesis of anxiety. Hence, searching for chemical agents with a multifunctional pharmacodynamic profile may bring highly effective therapy for CNS disorders. This study presents a deep structural insight into the hydantoin-arylpiperazine group and their serotonin/α-adrenergic activity. The newly synthesized compounds were tested in the radioligand binding assay and the intrinsic activity was evaluated for the selected derivatives. The computer-aided SAR analysis enabled us to answer questions about the influence of particular structural fragments on selective vs. multifunctional activity. As a result of the performed investigations, there were two leading structures: (a) compound 12 with multifunctional adrenergic-serotonin activity, which is a promising candidate to be an effective anxiolytic agent; (b) compound 14 with high α1A/α1D affinity and selectivity towards α1B, which is recommended due to the elimination of probable cardiotoxic effect. The structural conclusions of this work provide significant support for future lead optimization in order to achieve the desired pharmacodynamic profile in searching for new CNS-modulating agents.

Serotonin is a well-known neurotransmitter and neurohormone regulating mood, sleep, feeding, and learning in high organisms. Serotonin also affects the embryonic events related to neurogenesis and maturation of hormonal systems, the underlying organism adaptation to a changing environment. Such serotonin-based mother-to-embryo signaling is realized via direct interactions in case of internal fertilization and embryonic development inside the mother body. However, the possibility of such signaling is less obvious in organisms with the ancestral type of embryogenesis and embryo development within the egg, outside the mother body. Our data, based on the investigation of freshwater gastropod molluscs (Lymnaea and Helisoma), demonstrated a correlation between seasonal variations of serotonin content within the female reproductive system, and developmental patterns and the behavioral characteristics of progeny. The direct action of serotonin via posttranslational protein modification—serotonylation—during early development, as well as classical receptor-mediated effects, underlies such serotonin-modulated developmental changes. In the present paper, I will shortly overview our results on freshwater molluscs and parallel the experimental data with the living strategy of these species occupying almost all Holarctic regions.

Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2021-11-09T14:19:31Z No. of bitstreams: 2 Allana_Martins2021.pdf: 3215354 bytes, checksum: 6ae60b8076d22868214a2893b60cabc0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Made available in DSpace on 2021-11-09T14:19:31Z (GMT). No. of bitstreams: 2 Allana_Martins2021.pdf: 3215354 bytes, checksum: 6ae60b8076d22868214a2893b60cabc0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2021-09-03 Serotonin (5-HT) receptors are part of a family, the 5HT2 subfamily is divided into 2A, 2B and 2C receptors. The 2A receptor has high affinity for the atypical antipsychotic drugs clozapine, risperidone, and olanzapine and is believed to play a role in the therapeutic activity of these drugs. Currently, new therapeutic strategies are needed that offer faster onset of action with fewer side effects and, therefore, greater efficacy in a greater proportion of patients with neuropsychological disorders. Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction and communication, as well as the presence of restricted or repetitive behaviors and interests. Currently, several therapeutic targets have been described as associated with main or peripheral symptoms of this framework. However, so far, the few drug interventions aim to control certain associated symptoms, such as seizures, anxiety, irritation, lack of sleep, and others. In most cases, these are drugs commonly used in neuropsychiatry, such as anxiolytics, anticonvulsants, etc., used off-label, such as risperidone. Risperidone primarily targets the 5-HT2AR and D2R receptors, where it acts as an antagonist. However, this drug, although useful (or potentially useful), was originally and specifically developed for the treatment of autistic people. Thus, this work had as main objective to study a set of compounds selected from the literature, which were used in the identification of hit compounds possible candidates for antipsychotics that act as 5-HT2A receptor blockers. Based on these sets, computer-aided drug planning studies were carried out (molecular modeling, structure-activity relationship, pharmacophoric modeling and virtual screening), in addition to Molecular Dynamics simulation studies. The approach used led to the identification of compounds that were shown, in the DM simulations, to be stable at the binding site, capable of maintaining interactions with amino acid residues important for inhibition, similarly to risperidone. These results indicate a possibility that these compounds exhibit an antagonistic effect on the 5HT2A receptor. These compounds then become candidates for biological tests aiming at confirming the results obtained. Os receptores de serotonina (5-HT) fazem parte de uma família, a subfamília 5HT2 é dividida nos receptores 2A, 2B e 2C. O receptor 2A tem alta afinidade para os fármacos antipsicóticos atípicos clozapina, risperidona e olanzapina e acredita-se que tenha um papel na atividade terapêutica desses fármacos. Atualmente, são necessárias novas estratégias terapêuticas que ofereçam início de ação mais rápido com menos efeitos colaterais e, assim, maior eficácia em uma proporção maior de pacientes com distúrbios neuropsicológicos.O transtorno do espectro autista (TEA) é um distúrbio de neurodesenvolvimento caracterizado por déficits na interação social e comunicação, assim como a presença de comportamentos e interesses restritos ou repetitivos. Atualmente, vários alvos terapêuticos já foram descritos como associados a sintomas principais ou periféricos deste Quadro. Porém, até o momento, as poucas intervenções medicamentosas visam o controle de determinados sintomas associados, como convulsões, ansiedade, irritação, falta de sono, e outros. Na maioria dos casos, são medicamentos utilizados comumente na neuropsiquiatria, como ansiolíticos, anticonvulsivantes, etc, utilizados de modo off label, como a risperidona. A risperidona tem como alvos principais os receptores 5-HT2AR e D2R, locais onde age como antagonista. Porém, este fármaco, embora útil (ou potencialmente útil), foi originalmente e especificamente desenvolvido para o tratamento de pessoas autistas. Assim, este trabalho teve como objetivo principal estudar um conjunto de compostos selecionado da literatura, que foram empregados na identificação de compostos hits possíveis candidatos a antipsicóticos que atuam como bloqueadores do receptor 5-HT2A. A partir destes conjuntos, foram realizados estudos de planejamento de fármacos auxiliado com computador (modelagem molecular, relação estrutura atividade, modelagem farmacofórica e triagem virtual), além de estudos de simulação de Dinâmica Molecular (DM). A abordagem utilizada levou a identificação de compostos que se mostraram, nas simulações de DM, estáveis no sítio de ligação, capazes de manter as interações com os resíduos de aminoácidos importantes para a inibição, de modo similar a risperidona. Esses resultados indicam uma possibilidade desses compostos exibirem um efeito antagonista sobre o receptor 5HT2A. Estes compostos passam então a ser candidatos a ensaios biológicos visando a confirmação dos resultados obtidos.

BackgroundThe development of new non-surgical treatments dedicated to mitral valve degeneration is limited by the absence of relevant spontaneous and rapidly progressing animal experimental models.AnimalsWe characterized the spontaneous mitral valve degeneration in two inbred FVB mouse strains compared to C57BL/6J and investigated a contribution of the serotonergic system.MethodsMales and females FVB/NJ and FVB/NRj were compared to the putative C57BL/6J control at 12, 16, 20 and 24 weeks of age. Body weight, systolic blood pressure, heart rate, urinary 5-hydroxyindoleacetic acid (5-HIAA), whole blood and plasma serotonin, tail bleeding time, blood cell count, plasma TGF-β1 and plasma natriuretic peptide concentrations were measured. Myocardium and mitral valves were characterized by histology. mRNA mitral expression of 5-HT2A and 5-HT2B receptors was measured in the anterior leaflet. Cardiac anatomy and function were assessed by echocardiography.ResultsCompared to C57BL/6J, FVB mice strains did not significantly differ regarding body weight increase, arterial blood pressure and heart rate. A progressive augmentation of plasma pro-ANP was observed in FVB mice. Nevertheless, no cardiac hypertrophy or left-ventricular fibrosis were observed. Accordingly, plasma TGF-β1 was not different among the three strains. Conversely, FVB mice demonstrated a high prevalence of fibromyxoid highly cellularized and enriched in glycosaminoglycans lesions, inducing major mitral leaflets thickening without increase in length. The increased thickness was correlated with urinary 5-HIAA and blood platelet count. Whole blood serotonin concentration was similar in the two strains but, in FVB, a reduction of plasma serotonin was observed together with an increase of the bleeding time. Finally, echocardiography identified left atrial and left ventricular remodeling associated with thickening of both mitral leaflets and mitral insufficient in 30% of FVB mice but no systolic protrusion of mitral leaflets towards the atrium.ConclusionThe FVB mouse strain is highly prone to spontaneous mitral myxomatous degeneration. A contribution of the peripheral serotonergic system is suggested.

Short-lived turquoise killifish (Nothobranchius furzeri) have become a popular model organism for neuroscience. In the present paper we study for the first time their behavior in the novel tank diving test and the levels of mRNA of various 5-HT-related genes in brains of 2-, 4- and 6-month-old males and females of N. furzeri. The marked effect of age on body mass, locomotor activity and the mRNA level of Tph1b, Tph2, Slc6a4b, Mao, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes in the brains of N. furzeri males was shown. Locomotor activity and expression of the Mao gene increased, while expression of Tph1b, Tph2, Slc6a4b, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes decreased in 6-month-old killifish. Significant effects of sex on body mass as well as on mRNA level of Tph1a, Tph1b, Tph2, Slc6a4b, Htr1aa, 5-HT2a, Htr3a, Htr3b, Htr4, and Htr6 genes were revealed: in general both the body mass and the expression of these genes were higher in males. N. furzeri is a suitable model with which to study the fundamental problems of age-related alterations in various mRNA levels related with the brains 5-HT system.

The serotonin (5-hydroxytryptamine, 5-HT) signaling system is involved in a variety of physiological functions, including the control of cognition, reward, learning, memory, and vasoconstriction in vertebrates. Contrary to the extensive studies in the mammalian system, little is known about the molecular characteristics of the avian serotonin signaling network. In this study, we cloned and characterized the full-length cDNA of three serotonin receptor genes (HTR1B, HTR1E and HTR1F) in chicken pituitaries. Synteny analyses indicated that HTR1B, HTR1E and HTR1F were highly conserved across vertebrates. Cell-based luciferase reporter assays showed that the three chicken HTRs were functional, capable of binding their natural ligands (5-HT) or selective agonists (CP94253, BRL54443, and LY344864) and inhibiting intracellular cAMP production in a dose-dependent manner. Moreover, activation of these receptors could stimulate the MAPK/ERK signaling cascade. Quantitative real-time PCR analyses revealed that HTR1B, HTR1E and HTR1F were primarily expressed in various brain regions and the pituitary. In cultured chicken pituitary cells, we found that LY344864 could significantly inhibit the secretion of PRL stimulated by vasoactive intestinal peptide (VIP) or forskolin, revealing that HTR1F might be involved in the release of prolactin in chicken. Our findings provide insights into the molecular mechanism and facilitate a better understanding of the serotonergic modulation via HTR1B, HTR1E and HTR1F in avian species.

The study is dedicated to the investigation of serotonin (5-hydroxytryptamine, 5-HT) and 5-HT7 type serotonin receptor of localisation in larvae of two parasitic flatworms Opisthorchis felineus (Rivolta, 1884) Blanchard, 1895 and Hymenolepis diminuta Rudolphi, 1819, performed using the immunocytochemical method and confocal laser scanning microscopy (CLSM). Using whole mount preparations and specific antibodies, a microscopic analysis of the spatial distribution of 5-HT7-immunoreactivity(-IR) was revealed in worm tissue. In metacercariae of O. felineus 5-HT7-IR was observed in the main nerve cords and in the head commissure connecting the head ganglia. The presence of 5-HT7-IR was also found in several structures located on the oral sucker. 5-HT7-IR was evident in the round glandular cells scattered throughout the larva body. In cysticercoids of H. diminuta immunostaining to 5-HT7 was found in flame cells of the excretory system. Weak staining to 5-HT7 was observed along the longitudinal and transverse muscle fibres comprising the body wall and musculature of suckers, in thin longitudinal nerve cords and a connective commissure of the central nervous system. Available publications on serotonin action in flatworms and serotonin receptors identification were reviewed. Own results and the published data indicate that the muscular structures of flatworms are deeply supplied by 5-HT7-IR elements. It suggests that the 5-HT7 type receptor can mediate the serotonin action in the investigated species and is an important component of the flatworm motor control system. The study of the neurochemical basis of parasitic flatworms can play an important role in the solution of fundamental problems in early development of the nervous system and the evolution of neuronal signalling components.

Hallucinogens are a loosely defined group of compounds including LSD, N,N-dimethyltryptamines, mescaline, psilocybin/psilocin, and 2,5-dimethoxy-4-methamphetamine (DOM), which can evoke intense visual and emotional experiences. We are witnessing a renaissance of research interest in hallucinogens, driven by increasing awareness of their psychotherapeutic potential. As such, we now present a narrative review of the literature on hallucinogen binding in vitro and ex vivo, and the various molecular imaging studies with positron emission tomography (PET) or single photon emission computer tomography (SPECT). In general, molecular imaging can depict the uptake and binding distribution of labelled hallucinogenic compounds or their congeners in the brain, as was shown in an early PET study with N1-([11C]-methyl)-2-bromo-LSD ([11C]-MBL); displacement with the non-radioactive competitor ketanserin confirmed that the majority of [11C]-MBL specific binding was to serotonin 5-HT2A receptors. However, interactions at serotonin 5HT1A and other classes of receptors and pleotropic effects on second messenger pathways may contribute to the particular experiential phenomenologies of LSD and other hallucinogenic compounds. Other salient aspects of hallucinogen action include permeability to the blood–brain barrier, the rates of metabolism and elimination, and the formation of active metabolites. Despite the maturation of radiochemistry and molecular imaging in recent years, there has been only a handful of PET or SPECT studies of radiolabeled hallucinogens, most recently using the 5-HT2A/2C agonist N-(2[11CH3O]-methoxybenzyl)-2,5-dimethoxy- 4-bromophenethylamine ([11C]Cimbi-36). In addition to PET studies of target engagement at neuroreceptors and transporters, there is a small number of studies on the effects of hallucinogenic compounds on cerebral perfusion ([15O]-water) or metabolism ([18F]-fluorodeoxyglucose/FDG). There remains considerable scope for basic imaging research on the sites of interaction of hallucinogens and their cerebrometabolic effects; we expect that hybrid imaging with PET in conjunction with functional magnetic resonance imaging (fMRI) should provide especially useful for the next phase of this research., Molecules, 26 (9), ISSN:1420-3049

Ethnopharmacological relevance: The leaves and stems ofLippia multifloraMoldenke (Verbenaceae) are used in traditional medicine to treat depression, anxiety and epilepsy. Aim of the study:The present study investigated the anxiolytic-like effects ofLippia multifloraaqueous extracts in experimental models of anxiety. Materials and methods:Elevated plus maze and open field tests were used to evaluate anxiety and exploratory behaviour. The possible mechanism of action ofLippia multiflorawere investigated after pretreatment of animal with agonist and antagonist of serotonin receptors, such as: mCPP, a partial agonist at the 5-HT2Creceptors; Way-100635, a selective 5-HT1Areceptors antagonist; and DOI, the 5-HT2A/2Creceptors agonist. The levels of monoamine neurotransmitters were evaluated in the brain. Results:In the elevated plus maze,Lippia multifloraincreased the number of entries into and percentage of time in open arms, and reduced rearing, head dipping, and percentage of time in closed arms. These effects were blocked with the agonist and antagonist of serotonin receptors. In the open field,Lippia multifloraincreased crossing and reduced rearing and defecation. The endogenous levels of noradrenaline, dopamine and serotonin in the brain homogenates remain comparable to that distilled water-treated animals. Conclusion:It could be concluded thatLippia multifloraaqueous extracts, used in traditional medicine in Cameroon for the treatment of depression, anxiety and epilepsy seemed to possess anxiolytic properties in mice. These effects might be mediated by serotonin receptors, but not by modulation of the levels of monoamine neurotransmitters when the extract is administered once after one hour.

Introduction: The aim of this study was to determine the mRNA expression profile of dopamine D1, D2, D3, D4 and serotonin 5-HT1A, 5-HT2A, and 5-HT3A receptors in peripheral blood mononuclear cells (PBMCs) in schizophrenia and the in vitro effect of antipsychotics on the expression of these receptors in PBMCs of healthy subjects.Materials and Methods: Twenty-seven patients with schizophrenia and 29 healthy controls were recruited for the study. All study subjects underwent thorough clinical assessment, including anthropometric and body composition measurements. The expression of mRNA for dopamine D1−4 and serotonin 5-HT1A−3A receptors was measured using quantitative RT-PCR in peripheral blood mononuclear cells. In vitro mRNA and protein expression of these receptors was measured using quantitative RT-PCR and Western Blotting in PBMCs cultured with quetiapine, haloperidol, aripiprazole, risperidone, olanzapine or clozapine at IC50, half of IC50, and one-quarter of IC50 concentrations.Results: The key finding was that the schizophrenia group demonstrated significantly higher mRNA expression of D1, D2 and D4 receptors (p < 0.001), and significantly lower mRNA expression of 5-HT3A receptors (p < 0.01). After adjusting for smoking, the mRNA expression of D1 lost its significance, while that of D3, 5-HT1A, 5-HT2A became significant (all three were lower in the schizophrenia group). These receptors also demonstrated different ratios of mRNA expression in the schizophrenia group. The in vitro experiments showed that high concentrations of antipsychotics influenced the mRNA and protein expression of all studied receptors.Conclusion: Schizophrenia patients display a distinctive pattern of dopamine and serotonin receptor mRNA expression in blood mononuclear cells. This expression is little affected by antipsychotic treatment and it may therefore serve as a useful diagnostic biomarker for schizophrenia.

U ovom radu prikazana je povijesna primjena tvari iz skupine psihodelika, njihova glavna farmakološka obiljeţja temeljena na velikom broju pretkliničkih i kliničkih istraživanja te rezultati recentnih kliničkih studija. Klasični serotoninski psihodelici, vjerojatno djelovanjem preko 5-HT2A receptora, su pokazali pozitivan učinak u liječenju psihičkih oboljenja kao što su depresija, anksioznost, opsesivno-kompulzivni poremećaj, autizam, zatim ovisnosti poput alkoholizma i ovisnosti o nikotinu. U radu su prikazani neki temeljni, pretpostavljeni mehanizmi kojima psihodelici ostvaruju pozitivne terapijske učinke, kao i njihove prednosti i nedostaci u odnosu na konvencionalnu terapiju. Međutim, unatoč velikom napretku u istraživanjima, i dalje se nedovoljno zna o točnim mehanizmima terapijskog djelovanja psihodelika. Ostaje daljnjim istraživanjima ispitati točan farmakodinamski profil, kako bi se moglo točno odrediti koji psihodelik najbolje djeluje u kojoj indikaciji, u kojoj dozi ga je potrebno primijeniti te koji su rizici dugotrajne primjene, posebice za određene rizične skupine bolesnika. Ovim otkrićima otvoren je put ka razvoju novih učinkovitijih i sigurnijih terapijskih opcija različitih psihičkih bolesti, kako bi se oboljelima omogućila što bolja terapija te veća kvaliteta života. This paper presents historical use of substances from the class of psychedelics, their main pharmacological characteristics that are based on a large number of preclinical and clinical studies and the results of recent clinical studies. Classical serotonin psychedelics, presumably by acting via 5-HT2A receptors, have shown a positive effect in the treatment of mental illnesses such as depression, anxiety, obsessive-compulsive disorder, autism and addictions like alcoholism and nicotine addiction. The paper presents some basic, assumed mechanisms by which psychedelics achieve positive therapeutic effects, as well as their advantages and disadvantages compared to conventional therapy. However, despite great advances in research, knowledge about the exact mechanisms of therapeutic action of psychedelics is still insufficient. By further research it remains to examine the exact pharmacodynamic profile in order to determine exactly which psychedelic works best in which indication, in what dose it should be used and what are the risks of long-term use, especially for certain risk groups of patients. These discoveries paved the way for the development of new more effective and safer therapeutic options for various mental illnesses in order to provide patients with the best possible therapy and a higher quality of life.

Cestodes are platyhelminth parasites with a wide range of hosts that cause neglected diseases. Neurotransmitter signaling is of critical importance for these parasites which lack circulatory, respiratory and digestive systems. For example, serotonin (5-HT) and serotonergic G-protein coupled receptors (5-HT GPCRs) play major roles in cestode motility, development and reproduction. In previous work, we deorphanized a group of 5-HT7 type GPCRs from cestodes. However, little is known about another type of 5-HT GPCR, the 5-HT1 clade, which has been studied in several invertebrate phyla but not in platyhelminthes. Three putative 5-HT GPCRs from Echinococcus canadensis, Mesocestoides vogae (syn. M. corti) and Hymenolepis microstoma were cloned, sequenced and bioinformatically analyzed. Evidence grouped these new sequences within the 5-HT1 clade of GPCRs but differences in highly conserved GPCR motifs were observed. Transcriptomic analysis, heterologous expression and immunolocalization studies were performed to characterize the E. canadensis receptor, called Eca-5-HT1a. Functional heterologous expression studies showed that Eca-5-HT1a is highly specific for serotonin. 5-Methoxytryptamine and α-methylserotonin, both known 5-HT GPCR agonists, give stimulatory responses whereas methysergide, a known 5-HT GPCR ligand, give an antagonist response in Eca-5-HT1a. Mutants obtained by the substitution of key predicted residues resulted in severe impairment of receptor activity, confirming that indeed, these residues have important roles in receptor function. Immunolocalization studies on the protoscolex stage from E. canadensis, showed that Eca-5-HT1a is localized in branched fibers which correspond to the nervous system of the parasite. The patterns of immunoreactive fibers for Eca-5-HT1a and for serotonin were intimately intertwined but not identical, suggesting that they are two separate groups of fibers. These data provide the first functional, pharmacological and localization report of a serotonergic receptor that putatively belongs to the 5-HT1 type of GPCRs in cestodes. The serotonergic GPCR characterized here may represent a new target for antiparasitic intervention.

Serotonin receptors are extensively examined by academic and industrial researchers, due to their vital roles, which they play in the organism and constituting therefore important drug targets. Up to very recently, it was assumed that the basic nitrogen in compound structure is a necessary component to make it active within this receptor system. Such nitrogen interacts in its protonated form with the aspartic acid from the third transmembrane helix (D3x32) forming a hydrogen bond tightly fitting the ligand in the protein binding site. However, there are several recent studies that report strong serotonin receptor affinity also for compounds without a basic moiety in their structures. In the study, we carried out a comprehensive in silico analysis of the low-basicity phenomenon of the selected serotonin receptor ligands. We focused on the crystallized representatives of the proteins of 5-HT1B, 5-HT2A, 5-HT2B, and 5-HT2C receptors, and examined the problem both from the ligand- and structure-based perspectives. The study was performed for the native proteins, and for D3x32A mutants. The investigation resulted in the determination of nonstandard structural requirements for activity towards serotonin receptors, which can be used in the design of new nonbasic ligands.

G protein-coupled receptors are linked to various intracellular transducers, each pathway associated with different physiological effects. Biased ligands, capable of activating one pathway over another, are gaining attention for their therapeutic potential, as they could selectively activate beneficial pathways whilst avoiding those responsible for adverse effects. We performed molecular dynamics simulations with known &beta, arrestin-biased ligands like lysergic acid diethylamide and ergotamine in complex with the 5-HT2B receptor and discovered that the extent of ligand bias is directly connected with the degree of closure of the extracellular loop region. Given a loose allosteric coupling of extracellular and intracellular receptor regions, we delineate a concept for biased signaling at serotonin receptors, by which conformational interference with binding pocket closure restricts the signaling repertoire of the receptor. Molecular docking studies of biased ligands gathered from the BiasDB demonstrate that larger ligands only show plausible docking poses in the ergotamine-bound structure, highlighting the conformational constraints associated with bias. This emphasizes the importance of selecting the appropriate receptor conformation on which to base virtual screening workflows in structure-based drug design of biased ligands. As this mechanism of ligand bias has also been observed for muscarinic receptors, our studies provide a general mechanism of signaling bias transferable between aminergic receptors.

Five different subunits of the human serotonin 3 (5-hydroxytrptamine 3; 5-HT3) receptor exist and these are present in both central and peripheral systems. Different subunits alter the efficacy of 5-HT3 receptor antagonists used to treat diarrhoea predominant-irritable bowel syndrome, chemotherapy induced nausea and vomiting and depression. Cell surface arrangement of 5-HT3 receptor complexes and the contribution of C, D and E subunits to receptor function is poorly understood. Here, we examine interactions of A and C subunits using 5-HT3 receptor subunits containing fluorescent protein inserts between the 3rd and 4th transmembrane spanning region. HEK293T cells that do not normally express 5-HT3 receptor subunits, were transiently transfected with A or C or both subunits. Patch clamp experiments show that cells transfected with either fluorescent protein tagged A or A and C subunits generate whole cell currents in response to 5-HT. These findings correlate with the apparent distribution of fluorescent protein tagged A and C subunits at or near cell surfaces detected using TIRF microscopy. In co-transfected cells, the A and C subunits are associated forming AC heteromer complexes at or near the cell surface and a proportion can also form A or C homomers. In conclusion, it is likely that both A homomers and AC heteromers contribute to whole cell currents in response to 5-HT with minimal contribution from C homomers.

Calcific aortic valve disease (CAVD) is a deadly disease that is rising in prevalence due to population aging. While the disease is complex and poorly understood, one well-documented driver of valvulopathy is serotonin agonism. Both serotonin overexpression, as seen with carcinoid tumors and drug-related agonism, such as with Fenfluramine use, are linked with various diseases of the valves. Thus, the objective of this study was to determine if genetic ablation or pharmacological antagonism of the 5-HT2Bserotonin receptor (gene:Htr2b) could improve the hemodynamic and histological progression of calcific aortic valve disease.Htr2bmutant mice were crossed withNotch1+/-mice, an established small animal model of CAVD, to determine if genetic ablation affects CAVD progression. To assess the effect of pharmacological inhibition on CAVD progression,Notch1+/-mice were treated with the 5-HT2Breceptor antagonist SB204741. Mice were analyzed using echocardiography, histology, immunofluorescence, and real-time quantitative polymerase chain reaction.Htr2bmutant mice showed lower aortic valve peak velocity and mean pressure gradient–classical hemodynamic indicators of aortic valve stenosis–without concurrent left ventricle change. 5-HT2Breceptor antagonism, however, did not affect hemodynamic progression. Leaflet thickness, collagen density, and CAVD-associated transcriptional markers were not significantly different in any group. This study reveals that genetic ablation ofHtr2battenuates hemodynamic development of CAVD in theNotch1+/-mice, but pharmacological antagonism may require high doses or long-term treatment to slow progression.

Plant parasitic nematodes are microscopic pathogens that invade plant roots and cause extensive damage to crops. We have used a chemical biology approach to define mechanisms underpinning their parasitic behaviour: We discovered that reserpine, a plant alkaloid that inhibits the vesicular monoamine transporter (VMAT), potently impairs the ability of the potato cyst nematode Globodera pallida to enter the host plant root. We show this is due to an inhibition of serotonergic signalling that is essential for activation of the stylet which is used to access the host root. Prompted by this we identified core molecular components of G. pallida serotonin signalling encompassing the target of reserpine, VMAT; the synthetic enzyme for serotonin, tryptophan hydroxylase; the G protein coupled receptor SER-7 and the serotonin-gated chloride channel MOD-1. We cloned each of these molecular components and confirmed their functional identity by complementation of the corresponding C. elegans mutant thus mapping out serotonergic signalling in G. pallida. Complementary approaches testing the effect of chemical inhibitors of each of these signalling elements on discrete sub-behaviours required for parasitism and root invasion reinforce the critical role of serotonin. Thus, targeting the serotonin signalling pathway presents a promising new route to control plant parasitic nematodes., Author summary Indian snakeroot is an herbal medicine prepared from the roots of the shrub Rauwolfia serpentina that has been used for centuries for its calming action. We have found that the major active constituent of snakeroot, reserpine, is a potentially powerful crop protectant as it protects the roots of plants from attack by microscopic plant parasitic nematodes. The pharmacology of reserpine is well known: it works by depleting a specific class of mood regulating chemical in the nervous system, the biogenic amines. We show that in the nematode, reserpine disables biogenic amine signalling and that loss of the amine serotonin halts the parasitic life cycle. We identified key components of the serotonin signalling pathway in the potato cyst nematode Globodera pallida and show that chemicals that target these sites also inhibit the ability of the nematode to invade its host plant. Therefore, the naturally occurring plant root alkaloid reserpine has provided insight into new targets for crop protection.

Sensory systems rely on neuromodulators, such as serotonin, to provide flexibility for information processing as stimuli vary, such as light intensity throughout the day. Serotonergic neurons broadly innervate the optic ganglia of Drosophila melanogaster, a widely used model for studying vision. It remains unclear whether serotonin modulates the physiology of interneurons in the optic ganglia. To address this question, we first mapped the expression patterns of serotonin receptors in the visual system, focusing on a subset of cells with processes in the first optic ganglion, the lamina. Serotonin receptor expression was found in several types of columnar cells in the lamina including 5-HT2B in lamina monopolar cell L2, required for spatiotemporal luminance contrast, and both 5-HT1A and 5-HT1B in T1 cells, whose function is unknown. Subcellular mapping with GFP-tagged 5-HT2B and 5-HT1A constructs indicated that these receptors localize to layer M2 of the medulla, proximal to serotonergic boutons, suggesting that the medulla neuropil is the primary site of serotonergic regulation for these neurons. Exogenous serotonin increased basal intracellular calcium in L2 terminals in layer M2 and modestly decreased the duration of visually induced calcium transients in L2 neurons following repeated dark flashes, but otherwise did not alter the calcium transients. Flies without functional 5-HT2B failed to show an increase in basal calcium in response to serotonin. 5-HT2B mutants also failed to show a change in amplitude in their response to repeated light flashes but other calcium transient parameters were relatively unaffected. While we did not detect serotonin receptor expression in L1 neurons, they, like L2, underwent serotonin-induced changes in basal calcium, presumably via interactions with other cells. These data demonstrate that serotonin modulates the physiology of interneurons involved in early visual processing in Drosophila., Author summary Serotonergic neurons innervate the Drosophila melanogaster eye, but it was not known whether serotonin signaling could induce acute physiological responses in visual interneurons. We found serotonin receptors expressed in all neuropils of the optic lobe and identified specific neurons involved in visual information processing that express serotonin receptors. Activation of these receptors increased intracellular calcium in first order interneurons L1 and L2 and may enhance visually induced calcium transients in L2 neurons. These data support a role for the serotonergic neuromodulation of interneurons in the Drosophila visual system.

Chikungunya virus (CHIKV) is an important reemerging human pathogen transmitted by mosquitoes. The virus causes an acute febrile illness, chikungunya fever, which is characterized by headache, rash, and debilitating (poly)arthralgia that can reside for months to years after infection. Currently, effective antiviral therapies and vaccines are lacking. Due to the high morbidity and economic burden in the countries affected by CHIKV, there is a strong need for new strategies to inhibit CHIKV replication. The serotonergic drug 5-nonyloxytryptamine (5-NT) was previously identified as a potential host-directed inhibitor for CHIKV infection. In this study, we determined the mechanism of action by which the serotonin receptor agonist 5-NT controls CHIKV infection. Using time-of-addition and entry bypass assays, we found that 5-NT predominantly inhibits CHIKV in the early phases of the replication cycle, at a step prior to RNA translation and genome replication. Intriguingly, however, no effect was seen during virus-cell binding, internalization, membrane fusion and genomic RNA (gRNA) release into the cell cytosol. In addition, we show that the serotonin receptor antagonist methiothepin mesylate (MM) also has antiviral properties toward CHIKV and specifically interferes with the cell entry process and/or membrane fusion. Taken together, pharmacological targeting of 5-HT receptors may represent a potent way to limit viral spread and disease severity.IMPORTANCE The rapid spread of mosquito-borne viral diseases in humans puts a huge economic burden on developing countries. For many of these infections, including those caused by chikungunya virus (CHIKV), there are no specific treatment possibilities to alleviate disease symptoms. Understanding the virus-host interactions that are involved in the viral replication cycle is imperative for the rational design of therapeutic strategies. In this study, we discovered an antiviral compound, elucidated its mechanism of action, and propose serotonergic drugs as potential host-directed antivirals for CHIKV.

A total of 201 patients with major depressive disorder from four hospitals in Malaysia were followed up for 5 years to determine the prognostic factors of recurrent major depressive disorder that could potentially contribute to improving the management of MDD patients. For each individual patient, at the time of recruitment as part of a case-control study, information was collected on recent threatening life events, personality and social and occupational functioning, while blood samples were collected to genotype single nucleotide polymorphisms of vitamin D receptor (VDR), zinc transporter-3 (ZnT3), dopamine transporter-1 (DAT1), brain-derived neurotropic factor (BDNF), serotonin receptor 1A (HT1A) and 2A (HT2A) genes. Kaplan-Meier and Cox-regression were used to estimate hazard functions for recurrence of major depressive disorder. Individuals with severe MDD in previous major depressive episodes had five and a half times higher hazard of developing recurrence compared to mild and moderate MDD (HR = 5.565, 95% CI = 1.631-18.994, p = 0.006). Individuals who scored higher on social avoidance had three and a half times higher hazard of recurrence of MDD (HR = 3.525, 95% CI = 1.349-9.209; p = 0.010). There was significant interaction between ApaI +64978C>A single nucleotide polymorphism and severity. The hazard ratio increased by 6.4 times from mild and moderate to severe MDD for A/A genotype while that for C/A genotype increased by 11.3 times. Social avoidance and severity of depression at first episode were prognostic of recurrence. Screening for personality factors at first encounter with MDD patients needs to be considered as part of the clinical practice. For those at risk of recurrence in relation to social avoidance, the psychological intervention prescribed should be customized to focus on this modifiable factor. Prompt and appropriate management of severe MDD is recommended to reduce risk of recurrence.

Schizophrenia is a serious, chronic psychiatric disorder requiring lifelong treatment. Extrapyramidal side effects (EPS) are common adverse reactions to antipsychotic medications. In addition to the dopaminergic system, serotonergic mechanisms, including serotonin (5-HT) receptors, might be involved in EPS development. This study aimed to examine molecular associations of HTR1A, HTR1B, HTR2A, HTR2C and HTR6 gene polymorphisms with acute EPS in 229 male schizophrenia patients, following two weeks of haloperidol monotherapy. The Simpson&ndash, Angus Rating Scale for Extrapyramidal Side Effects (SAS), Barnes Akathisia Rating Scale (BARS) and Extrapyramidal Symptom Rating Scale (ESRS) were used to evaluate EPS severity. Genotyping was performed using real-time PCR, following extraction of blood DNA. Significant acute EPS appeared in 48.03% of schizophrenia patients. For the rs13212041 HTR1B gene polymorphism, affecting microRNA regulation of HTR1B gene expression, a higher frequency of TT carriers was found among haloperidol-treated patients with akathisia when compared to the group without akathisia symptoms. In comparison to C-allele carriers, patients carrying the TT genotype had higher akathisia severity, as determined by the SAS, BARS and ESRS scales. These molecular findings suggest potential involvement of 5-HT1B receptors in akathisia development following haloperidol treatment, as well as possible epigenetic mechanisms of serotonergic modulation associated with antipsychotic-induced EPS.

The multimodal antidepressant vortioxetine is thought to mediate its pharmacological effects via 5-HT 1A receptor agonism, 5-HT 1B receptor partial agonism, 5-HT 1D , 5-HT 3 , 5-HT 7 receptor antagonism and 5-HT transporter inhibition. Here we studied vortioxetine's functional effects across species (canine, mouse, rat, guinea pig and human) in cellular assays with heterologous expression of 5-HT 3A receptors (in Xenopus oocytes and HEK-293 cells) and in mouse neuroblastoma N1E-115 cells with endogenous expression of 5-HT 3A receptors. Furthermore, we studied the effects of vortioxetine on activity of CA1 Stratum Radiatum interneurons in rat hippocampus slices using current- and voltage-clamping methods. The patched neurons were subsequently filled with biocytin for confirmation of 5-HT 3 receptor mRNA expression by in situ hybridization. Whereas, both vortioxetine and the 5-HT 3 receptor antagonist ondansetron potently antagonized 5-HT-induced currents in the cellular assays, vortioxetine had a slower off-rate than ondansetron in oocytes expressing 5-HT 3A receptors. Furthermore, vortioxetine's but not ondansetron's 5-HT 3 receptor antagonistic potency varied considerably across species. Vortioxetine had the highest potency at rat and the lowest potency at guinea pig 5-HT 3A receptors. Finally, in 5-HT 3 receptor-expressing GABAergic interneurons from the CA1 stratum radiatum, vortioxetine and ondansetron blocked depolarizations induced by superfusion of either 5-HT or the 5-HT 3 receptor agonist mCPBG. Taken together, these data add to a growing literature supporting the idea that vortioxetine may inhibit GABAergic neurotransmission in some brain regions via a 5-HT 3 receptor antagonism-dependent mechanism and thereby disinhibit pyramidal neurons and enhance glutamatergic signaling.

Several studies confirmed the reciprocal interactions between adrenergic and serotoninergic systems and the influence of these phenomena on the pathogenesis of anxiety. Hence, searching for chemical agents with a multifunctional pharmacodynamic profile may bring highly effective therapy for CNS disorders. This study presents a deep structural insight into the hydantoin-arylpiperazine group and their serotonin/α-adrenergic activity. The newly synthesized compounds were tested in the radioligand binding assay and the intrinsic activity was evaluated for the selected derivatives. The computer-aided SAR analysis enabled us to answer questions about the influence of particular structural fragments on selective vs. multifunctional activity. As a result of the performed investigations, there were two leading structures: (a) compound 12 with multifunctional adrenergic-serotonin activity, which is a promising candidate to be an effective anxiolytic agent, (b) compound 14 with high α1A/α1D affinity and selectivity towards α1B, which is recommended due to the elimination of probable cardiotoxic effect. The structural conclusions of this work provide significant support for future lead optimization in order to achieve the desired pharmacodynamic profile in searching for new CNS-modulating agents.

Short-lived turquoise killifish (Nothobranchius furzeri) have become a popular model organism for neuroscience. In the present paper we study for the first time their behavior in the novel tank diving test and the levels of mRNA of various 5-HT-related genes in brains of 2-, 4- and 6-month-old males and females of N. furzeri. The marked effect of age on body mass, locomotor activity and the mRNA level of Tph1b, Tph2, Slc6a4b, Mao, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes in the brains of N. furzeri males was shown. Locomotor activity and expression of the Mao gene increased, while expression of Tph1b, Tph2, Slc6a4b, Htr1aa, Htr2a, Htr3a, Htr3b, Htr4, Htr6 genes decreased in 6-month-old killifish. Significant effects of sex on body mass as well as on mRNA level of Tph1a, Tph1b, Tph2, Slc6a4b, Htr1aa, 5-HT2a, Htr3a, Htr3b, Htr4, and Htr6 genes were revealed: in general both the body mass and the expression of these genes were higher in males. N. furzeri is a suitable model with which to study the fundamental problems of age-related alterations in various mRNA levels related with the brains 5-HT system.

Background: The treatment of schizophrenia is challenging due to the wide range of symptoms (positive, negative, cognitive) associated with the disease. Typical antipsychotics that antagonize D2 receptors are effective in treating positive symptoms, but extrapyramidal side-effects (EPS) are a common occurrence. Atypical antipsychotics targeting 5-HT2A and D2 receptors are more effective at treating cogni-tive and negative symptoms compared to typical antipsychotics, but these drugs also result in side-effects such as metabolic syndromes. Objective: To identify evidence in the literature that elucidates the pharmacological profile of aripiprazole. Methods: We searched PubMed for peer reviewed articles on aripiprazole and its clinical efficacy, side-effects, pharmacolo-gy, and effects in animal models of schizophrenia symptoms. Results: Aripiprazole is a newer atypical antipsychotic that displays a unique pharmacological profile, including partial D2 agonism and functionally selective properties. Aripiprazole is effective at treating the positive symptoms of schizophrenia and has the potential to treat negative and cognitive symptoms at least as well as other atypical antipsychotics. The drug has a favorable side-effect profile and has a low propensity to result in EPS or metabolic syndromes. Animal models of schizophrenia have been used to determine the efficacy of aripiprazole in symptom management. In these instanc-es, aripiprazole resulted in the reversal of deficits in extinction, pre-pulse inhibition, and social withdrawal. Because aripipra-zole requires a greater than 90% occupancy rate at D2 receptors to be clinically active and does not produce EPS, this suggests a functionally selective effect on intracel-lular signaling pathways. Conclusion: A combination of factors such as dopamine system stabilization via partial agonism, functional selectivity at D2 receptors, and serotonin-dopamine system interaction may contribute to the ability of aripiprazole to successfully manage schizophrenia symptoms. This review examines these mechanisms of action to further clarify the pharmacological actions of aripiprazole.

Purpose The aim of this study was to assess the potential involvement of a specific subtype of 5-hydroxytryptamine (5-HT), 5HT2 receptors in neurally-induced contractions of the human detrusor. Methods Contractile responses to electrical field stimulation (EFS) were examined in human isolated urinary bladder muscle strips. The potentiation of EFS-induced detrusor contraction was examined by adding cumulative concentrations of a 5-HT and 5-HT2 receptor agonist, α-methyl-serotonin (α-Me-5-HT) (1nM-100μM) in the presence or absence of a 5-HT2 antagonist, ketanserin (5-HT2A>5-HT2C) or naftopidil (5-HT2B>5-HT2A) (0.3-3μM). Results 5-HT and α-Me-5-HT potentiated EFS-induced contraction with a maximal effect (Emax) of 37.6% and 38.6%, respectively, and with pEC50 (negative logarithm of the concentration required for a half-maximal response to an agonist) values of 8.3 and 6.8, respectively. Neither ketanserin nor naftopidil at any concentration produced a rightward displacement of the α-Me-5-HT concentration response curve. Instead, the Emax of α-Me-5-HT increased in the presence of ketanserin at 0.3-1μM and in the presence of naftopidil at 1μM to 51% and 56%, respectively, while the Emax in the presence of vehicle alone was 36%. The highest concentration (3μM) of either drug, however, fully reversed the enhancement. Conclusions The potentiating effect of α-Me-5-HT on neurally-induced contraction of human urinary bladder muscle strips was not found to be mediated via any 5-HT2 receptor subtypes. The underlying mechanism for the enhancement of the α-Me-5-HT potentiating effect on detrusor contractility by ketanserin and naftopidil remains unknown; however, our results suggest that these drugs may be useful for treating contractile dysfunction of the detrusor, as manifested in conditions such as underactive bladder.

Reduced function of the serotonin transporter (SERT) is associated with increased susceptibility to anxiety and depression and with type-2 diabetes, which is especially true in older women. Preference for a "Western diet" (WD), enriched with saturated fat, cholesterol, and sugars, may aggravate these conditions. In previous studies, decreased glucose tolerance, central and peripheral inflammation, dyslipidemia, emotional, cognitive, and social abnormalities were reported in WD-fed young female mice. We investigated the metabolic, molecular, and behavioral changes associated with a 3-week-long dietary regime of either the WD or control diet in 12-month-old female mice with three different Sert genotypes: homozygous (Slc6a4) gene knockout (Sert(-/-): KO), heterozygous (Sert(+/-): HET), or wild-type mice (Sert(+/+): WT). In the WT-WD and KO-WD groups, but not in HET-WD-fed mice, most of changes induced by the WD paralleled those found in the younger mice, including brain overexpression of inflammatory marker Toll-like receptor 4 (Tlr4) and impaired hippocampus-dependent performance in the marble test. However, the 12-month-old female mice became obese. Control diet KO mice exhibited impaired hippocampal-dependent behaviors, increased brain expression of the serotonin receptors Htr2c and Htr1b, as well as increased Tlr4 and mitochondrial regulator, peroxisome proliferator-activated receptor gamma-coactivator-1a (Ppargc1a). Paradoxically, these, and other changes, were reversed in KO-WD mutants, suggesting a complex interplay between Sert deficiency and metabolic factors as well as potential compensatory molecular mechanisms that might be disrupted by the WD exposure. Most, but not all, of the changes in gene expression in the brain and liver of KO mice were not exhibited by the HET mice fed with either diet. Some of the WD-induced changes were similar in the KO-WD and HET-WD-fed mice, but the latter displayed a "rescued" phenotype in terms of diet-induced abnormalities in glucose tolerance, neuroinflammation, and hippocampus-dependent performance. Thus, complete versus partial Sert inactivation in aged mice results in distinct metabolic, molecular, and behavioral consequences in response to the WD. Our findings show that Sert(+/-) mice are resilient to certain environmental challenges and support the concept of heterosis as evolutionary adaptive mechanism.

Reduced function of the serotonin transporter (SERT) is associated with increased susceptibility to anxiety and depression and with type-2 diabetes, which is especially true in older women. Preference for a "Western diet" (WD), enriched with saturated fat, cholesterol, and sugars, may aggravate these conditions. In previous studies, decreased glucose tolerance, central and peripheral inflammation, dyslipidemia, emotional, cognitive, and social abnormalities were reported in WD-fed young female mice. We investigated the metabolic, molecular, and behavioral changes associated with a 3-week-long dietary regime of either the WD or control diet in 12-month-old female mice with three different Sert genotypes: homozygous (Slc6a4) gene knockout (Sert(-/-): KO), heterozygous (Sert(+/-): HET), or wild-type mice (Sert(+/+): WT). In the WT-WD and KO-WD groups, but not in HET-WD-fed mice, most of changes induced by the WD paralleled those found in the younger mice, including brain overexpression of inflammatory marker Toll-like receptor 4 (Tlr4) and impaired hippocampus-dependent performance in the marble test. However, the 12-month-old female mice became obese. Control diet KO mice exhibited impaired hippocampal-dependent behaviors, increased brain expression of the serotonin receptors Htr2c and Htr1b, as well as increased Tlr4 and mitochondrial regulator, peroxisome proliferator-activated receptor gamma-coactivator-1a (Ppargc1a). Paradoxically, these, and other changes, were reversed in KO-WD mutants, suggesting a complex interplay between Sert deficiency and metabolic factors as well as potential compensatory molecular mechanisms that might be disrupted by the WD exposure. Most, but not all, of the changes in gene expression in the brain and liver of KO mice were not exhibited by the HET mice fed with either diet. Some of the WD-induced changes were similar in the KO-WD and HET-WD-fed mice, but the latter displayed a "rescued" phenotype in terms of diet-induced abnormalities in glucose tolerance, neuroinflammation, and hippocampus-dependent performance. Thus, complete versus partial Sert inactivation in aged mice results in distinct metabolic, molecular, and behavioral consequences in response to the WD. Our findings show that Sert(+/-) mice are resilient to certain environmental challenges and support the concept of heterosis as evolutionary adaptive mechanism.

The olfactory pathway integrates the odor information required to generate correct behavioral responses. To address how changes of serotonin signaling in two contralaterally projecting, serotonin-immunoreactive deutocerebral neurons impacts key odorant attraction in Drosophila melanogaster, we selectively alter serotonin signaling using the serotonin transporter with mutated serotonin binding sites in these neurons and analyzed the consequence on odorant-guided food seeking. The expression of the mutated serotonin transporter selectively changed the odorant attraction in an odorant-specific manner. The shift in attraction was not influenced by more up-stream serotonergic mechanisms mediating behavioral inhibition. The expression of the mutated serotonin transporter in CSD neurons did not influence other behaviors associated with food seeking such as olfactory learning and memory or food consumption. We provide evidence that the change in the attraction by serotonin transporter function might be achieved by increased serotonin signaling and by different serotonin receptors. The 5-HT1B receptor positively regulated the attraction to low and negatively regulated the attraction to high concentrations of acetic acid. In contrast, 5-HT1A and 5-HT2A receptors negatively regulated the attraction in projection neurons to high acetic acid concentrations. These results provide insights into how serotonin signaling in two serotonergic neurons selectively regulates the behavioral response to key odorants during food seeking.

Reduced function of the serotonin transporter (SERT) is associated with increased susceptibility to anxiety and depression and with type-2 diabetes, which is especially true in older women. Preference for a “Western diet” (WD), enriched with saturated fat, cholesterol, and sugars, may aggravate these conditions. In previous studies, decreased glucose tolerance, central and peripheral inflammation, dyslipidemia, emotional, cognitive, and social abnormalities were reported in WD-fed young female mice. We investigated the metabolic, molecular, and behavioral changes associated with a 3-week-long dietary regime of either the WD or control diet in 12-month-old female mice with three different Sert genotypes: homozygous (Slc6a4) gene knockout (Sert−/−: KO), heterozygous (Sert+/−: HET), or wild-type mice (Sert+/+: WT). In the WT-WD and KO-WD groups, but not in HET-WD-fed mice, most of changes induced by the WD paralleled those found in the younger mice, including brain overexpression of inflammatory marker Toll-like receptor 4 (Tlr4) and impaired hippocampus-dependent performance in the marble test. However, the 12-month-old female mice became obese. Control diet KO mice exhibited impaired hippocampal-dependent behaviors, increased brain expression of the serotonin receptors Htr2c and Htr1b, as well as increased Tlr4 and mitochondrial regulator, peroxisome proliferator-activated receptor gamma-coactivator-1a (Ppargc1a). Paradoxically, these, and other changes, were reversed in KO-WD mutants, suggesting a complex interplay between Sert deficiency and metabolic factors as well as potential compensatory molecular mechanisms that might be disrupted by the WD exposure. Most, but not all, of the changes in gene expression in the brain and liver of KO mice were not exhibited by the HET mice fed with either diet. Some of the WD-induced changes were similar in the KO-WD and HET-WD-fed mice, but the latter displayed a “rescued” phenotype in terms of diet-induced abnormalities in glucose tolerance, neuroinflammation, and hippocampus-dependent performance. Thus, complete versus partial Sert inactivation in aged mice results in distinct metabolic, molecular, and behavioral consequences in response to the WD. Our findings show that Sert+/− mice are resilient to certain environmental challenges and support the concept of heterosis as evolutionary adaptive mechanism. © Copyright © 2020 Veniaminova, Cespuglio, Chernukha, Schmitt-Boehrer, Morozov, Kalueff, Kuznetsova, Anthony, Lesch and Strekalova. 01EW1602B, 01EW1902, 602805 Deutsche Forschungsgemeinschaft, DFG: CRC TRR 58 A1/A5 Horizon 2020 Framework Programme, H2020: Eat2beNICE, 728018, 643051 Russian Science Foundation, RSF: 16-16-10073 Seventh Framework Programme, FP7 I.M. Sechenov First Moscow State Medical University, MSMU Deutsche Forschungsgemeinschaft, DFG We thank Collette Rousset and Donia Amrouni from C. Bernard University, Anna Gorlova from Sechenov University, and Anastasiya Kibitkina and Galina Tolmacheva from the Gorbatov Research Center for their valuable contribution to this work. Funding. The authors? work reported here was supported by the Deutsche Forschungsgemeinschaft (DFG: CRC TRR 58 A1/A5), the European Union?s Seventh Framework Programme (FP7/2007?2013) under Grant No. 602805 (Aggressotype), ERA-Net NEURON/RESPOND, No. 01EW1602B and ERA-Net NEURON/DECODE, No. 01EW1902 (to K-PL), the Horizon 2020 Research and Innovation Programme under Grant No. 728018 (Eat2beNICE) and Grant No. 643051 (MiND), 5-100 Russian Academic Excellence Project (to K-PL and TS), the Russian Science Foundation (RSF) Project No. 16-16-10073 (to IC and OK), and the University of W?rzburg in the funding programme Open Access Publishing (to KP-L).

The myriad conformations of a G protein-coupled receptors induced by ligands are critical for determining signaling cascades associated with distinct functional and behavioral consequences. For example, 5-hydroxytryptamine 2A receptors (5-HT2AR) are the target for classic hallucinogens, atypical antipsychotics, and plasticity-promoting psychoplastogens. However, current methods are inadequate for directly assessing specific 5-HT2AR conformations. Here, we developed psychLight, a genetically-encoded fluorescent sensor based on the 5-HT2AR structure. PsychLight reports behaviorally-relevant serotonin release and 5-HT2AR conformations induced by serotonergic hallucinogens. Using a stable cell line expressing psychLight, we correctly predicted the hallucinogenic behavioral effects of compounds even with structural similarity and with previously unknown hallucinogenic potentials. We further used psychLight to identify a novel, non-hallucinogenic psychedelic analog, which promoted structural neural plasticity and produced rapid- and long-lasting antidepressant-like effects after a single administration. The advent of psychLight will enable early identification of abusive designer drugs and the development of 5-HT2AR-dependent non-hallucinogenic therapeutics.

G Protein-Coupled Receptors (GPCRs) mediate intracellular signaling in response to extracellular ligand binding and are the target of one-third of approved drugs. Ligand binding modulates the GPCR molecular free energy landscape by preferentially stabilizing active or inactive conformations that dictate intracellular protein recruitment and downstream signaling. We perform enhanced sampling molecular dynamics simulations to recover the free energy surfaces of a thermostable mutant of the GPCR serotonin receptor 5-HT2B in the unliganded form and bound to a lysergic acid diethylamide (LSD) agonist and lisuride antagonist. LSD binding imparts a ∼110 kJ/mol driving force for conformational rearrangement into an active state. The lisuride-bound form is structurally similar to the apo form and only ∼24 kJ/mol more stable. This work quantifies ligand-induced conformational specificity and functional selectivity of 5-HT2B and presents a platform for high-throughput virtual screening of ligands and rational engineering of the ligand-bound molecular free energy landscape.

Opioids are the gold standard drugs for the treatment of acute and chronic severe pain, although their serious side effects constitute a big limitation. In the search for new and safer drugs, 5-HT1AR agonists are emerging as potential candidates in pain relief therapy. In this work, we evaluated the affinity and activity of enantiomers of the two newly synthesized, potent 5-HT1AR agonists N-[(2,2-diphenyl-1,3-dioxolan-4-yl)methyl]-2-[2-(pyridin-4-yl)phenoxy]ethan-1-ammonium hydrogenoxalate (rac-1) and N-((2,2-diphenyl-1,3-dioxolan-4-yl)methyl)-2-(2-(1-methyl-1H-imidazol-5-yl)phenoxy)ethan-1-ammonium hydrogenoxalate (rac-2) in vitro and in vivo. The role of chirality in the interaction with 5-HT1AR was evaluated by molecular docking. The activity of the rac-1 was tested in mouse models of acute pain (hot plate) and severe tonic nociceptive stimulation (intraplantar formalin test). Rac-1 was active in the formalin test with a reduction in paw licking in both phases at 10 mg/kg, and its effect was abolished by the selective 5-HT1AR antagonist, WAY-100635. The eutomer (S)-1, but not the racemate, was active during the hot plate test at 10 and 20 mg/kg, and this effect was abolished by 30 min treatment with WAY-100635 at 30 min. Similarly to 8-OH-DPAT, (S)-1 evoked a slow outward current and depressed spontaneous glutamatergic transmission in superficial dorsal horn neurons, more effectively than rac-1. The eutomer (S)-1 showed promising developability properties, such as high selectivity over 5-HT subtypes, no interaction with the μ receptors, and low hepato- and cardiotoxicity. Therefore, (S)-1 may represent a potential candidate for the treatment of acute and chronic pain without having the adverse effects that are commonly associated with the classic opioid drugs.

Reduced function of the serotonin transporter (SERT) is associated with increased susceptibility to anxiety and depression and with type-2 diabetes, which is especially true in older women. Preference for a “Western diet” (WD), enriched with saturated fat, cholesterol, and sugars, may aggravate these conditions. In previous studies, decreased glucose tolerance, central and peripheral inflammation, dyslipidemia, emotional, cognitive, and social abnormalities were reported in WD-fed young female mice. We investigated the metabolic, molecular, and behavioral changes associated with a 3-week-long dietary regime of either the WD or control diet in 12-month-old female mice with three different Sert genotypes: homozygous (Slc6a4) gene knockout (Sert−/−: KO), heterozygous (Sert+/−: HET), or wild-type mice (Sert+/+: WT). In the WT-WD and KO-WD groups, but not in HET-WD-fed mice, most of changes induced by the WD paralleled those found in the younger mice, including brain overexpression of inflammatory marker Toll-like receptor 4 (Tlr4) and impaired hippocampus-dependent performance in the marble test. However, the 12-month-old female mice became obese. Control diet KO mice exhibited impaired hippocampal-dependent behaviors, increased brain expression of the serotonin receptors Htr2c and Htr1b, as well as increased Tlr4 and mitochondrial regulator, peroxisome proliferator-activated receptor gamma-coactivator-1a (Ppargc1a). Paradoxically, these, and other changes, were reversed in KO-WD mutants, suggesting a complex interplay between Sert deficiency and metabolic factors as well as potential compensatory molecular mechanisms that might be disrupted by the WD exposure. Most, but not all, of the changes in gene expression in the brain and liver of KO mice were not exhibited by the HET mice fed with either diet. Some of the WD-induced changes were similar in the KO-WD and HET-WD-fed mice, but the latter displayed a “rescued” phenotype in terms of diet-induced abnormalities in glucose tolerance, neuroinflammation, and hippocampus-dependent performance. Thus, complete versus partial Sert inactivation in aged mice results in distinct metabolic, molecular, and behavioral consequences in response to the WD. Our findings show that Sert+/− mice are resilient to certain environmental challenges and support the concept of heterosis as evolutionary adaptive mechanism.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES Introduction: The Interferential current (IC) is an electrotherapeutic modality widely used for analgesic purposes, however, few studies have sought to evaluate its mechanisms of action to promote the antinociceptive effect. The aim of this study was to investigate the involvement of spinal serotonergic and noradrenergic receptors in the antinociceptive effect of the interferential current in the animal model of articular inflammation in Wistar rats. Material and method: We included in this study 54 male Wistar rats distributed in the groups CI + saline, CI + DMSO 20%, inactive IC + saline, inactive IC + DMSO 20%, CI + methysergide, CI + tropanil dichlorobenzoate, CI + ioimbine, serotonin hydrochloride and hydrochloride of clonidine. At baseline, behavioral tests were performed for sensitivity and motor performance followed by induction of joint inflammation by injection of 3% carragein solution and 3% caolin in the left knee of the animals. After 24 h, the behavior was reevaluated followed by treatment with the current, preceded or not by pharmacological blockade. The pharmacological blockade was performed by means of intrathecal injection, applied before the treatment with the current. One hours after treatment with IC, the behavioral tests were repeated. The values of p < 0.05 were considered significant. Results: There was a significant decrease in the mechanical threshold of left paw withdrawal 24 h after the induction of inflammation in all groups (p < 0.0001). The groups treated with interferential current that received vehicles (saline or DMSO 20%) Significantly increased the mechanical threshold of paw withdrawal in comparison to the pre-treatment moment (p ≤ 0.001). Both with very large effect size according to Cohen's d (d = 1.95 and d = 1.83, respectively). There was blockade of the effect of IC in the groups that received, prior to the treatment with the current, the methysergide (non-selective antagonist of receptors 5-HT1 and 5-HT2) and tropanyl dichlorobenzoate (receptor antagonist 5-HT3). In these two groups, there was no statistically significant difference in the mechanical threshold of left paw withdrawal, when comparing the moments before and after treatment. In addition, after treatment, the pre-treated groups with methysergide or tropanyl dichlorobenzoate did not increase the mechanical threshold of left paw withdrawal, different from the vehicle groups. The animals that received yohimbine (α2-adrenergic antagonist) increased the mechanical threshold for the removal of the inflamed paw when comparing the pre-and post-treatment moments, showing that this pharmacological blockade did not prevent the effect of IC. Intrathecal injection of serotonergic and α2-adrenergic receptor agonists (serotonin hydrochloride and clonidine) significantly increased the mechanical threshold of left paw withdrawal (p ≤ 0.004). The distance traveled by the animals reduced significantly (p ≤ 0.0207) after the induction of inflammation, however, the administration of the drugs and/or treatment with IC did not exert influence on motor activity. Conclusion: This study shows part of the mechanism of action of IC in motor intensity to promote antinociceptive effect. This effect is mediated by spinal activation of 5-HT1, 5-HT2 and 5-HT3 receptors, but not α2-adrenergic receptors. These results allow professionals to justify the clinical indication of this electrotherapeutic resource, adapting its use to the physiopathological particularities of each individual. Introdução: A corrente interferencial (CI) é uma modalidade eletroterapêutica bastante utilizada para fins analgésicos, contudo, poucos estudos buscaram avaliar seus mecanismos de ação para promoção do efeito antinociceptivo. O objetivo deste estudo foi investigar a participação de receptores serotoninérgicos e noradrenérgicos espinais no efeito antinociceptivo da corrente interferencial no modelo animal de inflamação articular em ratos Wistar. Material e método: Foram incluídos neste estudo 54 ratos Wistar machos distribuídos nos grupos CI + salina, CI + DMSO 20%, CI inativa + salina, CI inativa + DMSO 20%, CI + metisergida, CI + tropanil diclorobenzoato, CI + ioimbina, cloridrato de serotonina e cloridrato de clonidina. No momento basal, foram realizados testes comportamentais para sensibilidade e desempenho motor seguido de indução de inflamação articular por injeção de solução de carragenina 3% e caolina 3% no joelho esquerdo dos animais. Após 24 h, o comportamento foi reavaliado seguido de tratamento com a corrente, antecedido ou não de bloqueio farmacológico. O bloqueio farmacológico foi realizado por meio de injeção intratecal, aplicada antes do tratamento com a corrente. Uma hora após o tratamento com a CI, os testes comportamentais foram repetidos. Os valores de p < 0,05 foram considerados significativos. Resultados: Houve diminuição significativa do limiar mecânico de retirada da pata esquerda 24 h após a indução da inflamação em todos os grupos (p < 0,0001). Os grupos tratados com corrente interferencial que receberam veículos (salina ou DMSO 20%) aumentaram significativamente o limiar mecânico de retirada da pata em comparação ao momento pré-tratamento (p ≤ 0,001). Ambos com tamanho de efeito muito grande de acordo com o d de Cohen (d = 1,95 e d = 1,83, respectivamente). Houve bloqueio do efeito da CI nos grupos que receberam, previamente ao tratamento com a corrente, a metisergida (antagonista não seletivo de receptores 5-HT1 e 5-HT2) e tropanil diclorobenzoato (antagonista de receptores 5-HT3). Nesses dois grupos, não houve diferença estatisticamente significante no limiar mecânico de retirada da pata esquerda, quando comparados os momentos pré e pós-tratamento. Além disso, no pós-tratamento, os grupos pré-tratados com metisergida ou tropanil diclorobenzoato não aumentaram o limiar mecânico de retirada da pata esquerda, diferente dos grupos veículos. Os animais que receberam ioimbina (antagonista α2-adrenérgico) aumentaram o limiar mecânico de retirada da pata inflamada quando comparados os momentos pré e pós-tratamento, mostrando que esse bloqueio farmacológico não impediu o efeito da CI. A injeção intratecal de agonistas de receptores serotoninérgicos e α2-adrenérgicos (cloridrato de serotonina e clonidina) aumentou significativamente o limiar mecânico de retirada da pata esquerda (p ≤ 0,004). A distância percorrida pelos animais reduziu significativamente (p ≤ 0,0207) após a indução da inflamação, contudo, a administração dos fármacos e/ou tratamento com a CI não exerceu influencia na atividade motora. Conclusão: Esse estudo mostra parte do mecanismo de ação da CI em intensidade motora para promoção de efeito antinociceptivo. Tal efeito é mediado por ativação espinal de receptores 5-HT1, 5-HT2 e 5-HT3, mas não de receptores α2-adrenérgicos. Esses resultados permitem aos profissionais justificar a indicação clínica desse recurso eletroterapêutico, adequando seu uso às particularidades fisiopatológicas de cada indivíduo. São Cristóvão, SE