The family of serotonin 5-HT2 receptors stimulates the phospholipase C second messenger pathway via the α subunit of the Gq GTP-binding protein. Here, we show that agonist stimulation of the 5-HT2B ...receptor subtype stably expressed in the mouse fibroblast LMTK− cell line causes a rapid and transient activation of the proto-oncogene product p21ras as measured by an increase in GTP-bound Ras in response to serotonin. Furthermore, 5-HT2B receptor stimulation activates p42mapk/p44mapk (ERK2/ERK1) mitogen-activated protein kinases as assayed by phosphorylation of myelin basic protein. Antibodies against p21ras, Gαq, -β, or -γ2 subunits of the GTP-binding protein inhibit MAP kinase-dependent phosphorylation. The MAP kinase activation is correlated with a stimulation of cell division by serotonin. In addition to this mitogenic action, transforming activity of serotonin is mediated by the 5-HT2B receptor since its expression in LMTK− cells is absolutely required for foci formation and for these foci to form tumors in nude mice. Finally, we detected expression of the 5-HT2B receptor in spontaneous human and Mastomys natalensis carcinoid tumors and, similar to the 5-HT2B receptor transfected cells, the Mastomys tumor cells are also responsive to serotonin with similar coupling to p21ras activation.
Objectives : Cocaine dependence has a strong heritability component. The aim of this study was to investigate the putative association between the serotonin 2B receptor gene (HTR2B), crack use ...disorders and impulsivity. Methods : A French Caribbean male population of patients with crack use disorders (n=80) w as compared to healthy male controls (n=60). Comorbid ADHD and impulsivity were assessed. Five Single Nucleotide Polymorphisms (SNPs) in the HTR2B gene on chromosome 2 were selected: rs643700, rs 6736017, rs1549339, rs17586428 and rs3806545 Thes e five SNPs were chosen to cover most of the linkage disequilibrium (LD) blocks in HTR2B. The French translation of the Baratt Impulsivity Scale BIS -11 was used to evaluate impulsivity. Comorbid ADHD was diagnosed using the Wender Utah Rating Scale-25 item for the Attention Deficit-Hyperactivity Disorder. Results : We have found a positive association between the rs6736017 polymorphism and crack use disorders in a French Afro-caribbean male population. Conclusion : The risk effect of HTR2B rs6736017 appeare d to be specific to individuals who are crack users rather than being driven by impulsivity alone or ADHD alone
Cannabis use is increasing in the United States, as are its adverse effects. We investigated the genetics of an adverse consequence of cannabis use: cannabis-related aggression (CRA) using a ...genome-wide association study (GWAS) design. Our GWAS sample included 3269 African Americans (AAs) and 2546 European Americans (EAs). An additional 89 AA subjects from the Grady Trauma Project (GTP) were also examined using a proxy-phenotype replication approach. We identified genome-wide significant risk loci contributing to CRA in AAs at the serotonin receptor 2B receptor gene (HTR2B), and the lead SNP, HTR2B*rs17440378, showed nominal association to aggression in the GTP cohort of cannabis-exposed subjects. A priori evidence linked HTR2B to impulsivity/aggression but not to cannabis response. Human functional data regarding the HTR2B variant further supported our finding. Treating an Htr2b.sup.-/- knockout mouse with THC resulted in increased aggressive behavior, whereas wild-type mice following THC administration showed decreased aggression in the resident-intruder paradigm, demonstrating that HTR2B variation moderates the effects of cannabis on aggression. These concordant findings in mice and humans implicate HTR2B as a major locus associated with cannabis-induced aggression.
The serotonin receptor subtypes 2 comprise 5-HT
, 5-HT
, and 5-HT
, which are Gα
-coupled receptors and display distinct pharmacological properties. Although co-expressed in some brain regions and ...involved in various neurological disorders, their functional interactions have not yet been studied. We report that 5-HT
receptors can form homo- and heterodimers when expressed alone or co-expressed in transfected cells. Co-immunoprecipitation and bioluminescence resonance energy transfer studies confirmed that 5-HT
receptors interact with either 5-HT
or 5-HT
receptors. Although heterodimerization with 5-HT
receptors does not alter 5-HT
Gα
-dependent inositol phosphate signaling, 5-HT
or 5-HT
receptor-mediated signaling was totally blunted. This feature can be explained by a dominance of 5-HT
on 5-HT
and 5-HT
receptor binding; in 5-HT
-containing heterodimers, ligands bind and activate the 5-HT
protomer exclusively. This dominant effect on the associated protomer was also observed in neurons, supporting the physiological relevance of 5-HT
receptor heterodimerization
Accordingly, exogenous expression of an inactive form of the 5-HT
receptor in the locus ceruleus is associated with decreased 5-HT
-dependent noradrenergic transmission. These data demonstrate that 5-HT
receptors can form functionally asymmetric heterodimers
and
that must be considered when analyzing the physiological or pathophysiological roles of serotonin in tissues where 5-HT
receptors are co-expressed.
The serotonin 2B (5-HT2B) receptor coupled to Gq-protein contributes to the control of neuronal excitability and is implicated in various psychiatric disorders. The mechanisms underlying its brain ...function are not fully described. Using peptide affinity chromatography combined with mass spectrometry, we found that the PDZ binding motif of the 5-HT2B receptor located at its C-terminal end interacts with the scaffolding protein channel interacting PDZ protein (CIPP). We then showed, in COS-7 cells, that the association of the 5-HT2B receptor to CIPP enhanced receptor-operated inositol phosphate (IP) production without affecting its cell surface and intracellular levels. Co-immunoprecipitation experiments revealed that CIPP, the 5-HT2B receptor, and the NR1 subunit of the NMDA receptor form a macromolecular complex. CIPP increased 5-HT2B receptor clustering at the surface of primary cultured hippocampal neurons and prevented receptor dispersion following agonist stimulation, thus potentiating IP production and intracellular calcium mobilization in dendrites. CIPP or 5-HT2B receptor stimulation in turn dispersed NR1 clusters colocalized with 5-HT2B receptors and increased the density and maturation of dendritic spines. Collectively, our results suggest that the 5-HT2B receptor, the NMDA receptor, and CIPP may form a signaling platform by which serotonin can influence structural plasticity of excitatory glutamatergic synapses.
Background and Purpose
Valvular heart disease (VHD) is highly prevalent in industrialized countries. Chronic use of anorexigens, amphetamine or ergot derivatives targeting the 5‐HT system is ...associated with VHD. Here, we investigated the contribution of 5‐HT receptors in a model of valve degeneration induced by nordexfenfluramine, the main metabolite of the anorexigens, dexfenfluramine and benfluorex.
Experimental Approach
Nordexfenfluramine was infused chronically (28 days) in mice ((WT and transgenic Htr2B ‐/‐, Htr2A ‐/‐, and Htr2B/2A ‐/‐) to induce mitral valve lesions. Bone marrow transplantation was also carried out. Haemodynamics were measured with echocardiography; tissues and cells were analysed by histology, immunocytochemistry, flow cytometry and RT –qPCR. Samples of human prolapsed mitral valves were also analysed.
Key Results
Chronic treatment of mice with nordexfenfluramine activated 5‐HT2B receptors and increased valve thickness and cell density in a thick extracellular matrix, mimicking early steps of mitral valve remodelling. Lesions were prevented by 5‐HT2A or 5‐HT2B receptor antagonists and in transgenic Htr2B −/− or Htr2A/2B −/− mice. Surprisingly, valve lesions were mainly formed by numerous non‐proliferative CD34+ endothelial progenitors. These progenitors originated from bone marrow (BM) as revealed by BM transplantation. The initial steps of mitral valve remodelling involved mobilization of BM‐derived CD34+CD31+ cells by 5‐HT2B receptor stimulation. Analysis of human prolapsed mitral valves showing spontaneous degenerative lesions, demonstrated the presence of non‐proliferating CD34+/CD309+/NOS3+ endothelial progenitors expressing 5‐HT2B receptors.
Conclusions and Implications
BM‐derived endothelial progenitor cells make a crucial contribution to the remodelling of mitral valve tissue. Our data describe a new and important mechanism underlying human VHD.
Cannabis use is increasing in the United States, as are its adverse effects. We investigated the genetics of an adverse consequence of cannabis use: cannabis-related aggression (CRA) using a ...genome-wide association study (GWAS) design. Our GWAS sample included 3269 African Americans (AAs) and 2546 European Americans (EAs). An additional 89 AA subjects from the Grady Trauma Project (GTP) were also examined using a proxy-phenotype replication approach. We identified genome-wide significant risk loci contributing to CRA in AAs at the serotonin receptor 2B receptor gene (HTR2B), and the lead SNP, HTR2B*rs17440378, showed nominal association to aggression in the GTP cohort of cannabis-exposed subjects. A priori evidence linked HTR2B to impulsivity/aggression but not to cannabis response. Human functional data regarding the HTR2B variant further supported our finding. Treating an Htr2b
knockout mouse with THC resulted in increased aggressive behavior, whereas wild-type mice following THC administration showed decreased aggression in the resident-intruder paradigm, demonstrating that HTR2B variation moderates the effects of cannabis on aggression. These concordant findings in mice and humans implicate HTR2B as a major locus associated with cannabis-induced aggression.
The serotonin (5-HT) syndrome occurs in humans after antidepressant overdose or combination of drugs inducing a massive increase in extracellular 5-HT. Several 5-HT receptors are known to participate ...in this syndrome in humans and animal models. The 5-HT(2B) receptor has been proposed as a positive modulator of serotonergic activity, but whether it is involved in 5-HT syndrome has not yet been studied. We analyzed here, a putative role of 5-HT(2B) receptors in this disorder by forced swimming test (FST) and behavioral assessment in the open field. In FST, genetic (5-HT(2B)(-/-) mice) or pharmacological (antagonist RS127445 at 0.5 mg/kg) ablation of 5-HT(2B) receptors facilitated selective 5-HT reuptake inhibitors (SSRI)-induced increase of immobility time as well as expression of other symptoms related to 5-HT syndrome like hind limb abduction and Straub tail. Increase in immobility was also developed in FST by both wild type (WT) and 5-HT(2B)(-/-) mice after the administration of 5-HT(1A), 5-HT(2A) or 5-HT(2C) receptor agonists, 8-OH-DPAT (5 mg/kg), DOI (1 mg/kg), or WAY161503 (5 mg/kg), respectively. In contrast, the 5-HT(2B) receptor agonist BW723C86 (3 mg/kg) or 5-HT(1B) receptor agonist CGS12066A (2 mg/kg) decreased immobility time in both genotypes. The 5-HT syndrome induced by fluoxetine at high doses was blocked in WT and 5-HT(2B)(-/-) mice by administration of 5-HT(1A) and 5-HT(2C) receptor antagonists (WAY100635 0.5 mg/kg and SB242084 0.5 mg/kg) but not by the 5-HT(2A) receptor antagonist MDL100907 (1 mg/kg). By behavioral assessment, we confirmed that 5-HT(2B)(-/-) mice were more prone to develop 5-HT syndrome symptoms after administration of high dose of SSRIs or the 5-HT precursor 5-Hydroxytryptophan, 5-HTP, even if increases in 5-HT plasma levels were similar in both genotypes. This evidence suggests that the presence of 5-HT(2B) receptors hinders acute 5-HT toxicity once high levels of 5-HT are attained. Therefore, differential agonism/antagonism of 5-HT receptors should be considered in the search of therapeutic targets for treating this serious disorder.
Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological ...changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called "directional motility", toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine 5-HT). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.
The "club drug" 3,4-methylenedioxymethamphetamine (MDMA; also known as ecstasy) binds preferentially to and reverses the activity of the serotonin transporter, causing release of serotonin ...5-hydroxytryptamine (5-HT) stores from nerve terminals. Subsequent activation of postsynaptic 5-HT receptors by released 5-HT has been shown to be critical for the unique psychostimulatory effects of MDMA. In contrast, the effects of direct activation of presynaptic and/or postsynaptic receptors by MDMA have received far less attention, despite the agonist actions of the drug itself at 5-HT(2) receptors, in particular the 5-HT(2B) receptor. Here we show that acute pharmacological inhibition or genetic ablation of the 5-HT(2B) receptor in mice completely abolishes MDMA-induced hyperlocomotion and 5-HT release in nucleus accumbens and ventral tegmental area. Furthermore, the 5-HT(2B) receptor dependence of MDMA-stimulated release of endogenous 5-HT from superfused midbrain synaptosomes suggests that 5-HT(2B) receptors act, unlike any other 5-HT receptor, presynaptically to affect MDMA-stimulated 5-HT release. Thus, our findings reveal a novel regulatory component in the actions of MDMA and represent the first demonstration that 5-HT(2B) receptors play an important role in the brain, i.e., modulation of 5-HT release. As such, 5-HT(2B) receptor antagonists may serve as promising therapeutic drugs for MDMA abuse.
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