Serotonin 2C Research Articles

Serotonin (2C) receptor regulation of cocaine-induced conditioned place preference and locomotor sensitization

Previous studies have identified an inhibitory regulatory role of the 5-HT2C receptor in serotonin and dopamine neurotransmission. As cocaine is known to enhance serotonin and dopamine transmission, the ability of 5-HT2C receptors to modulate cocaine-induced behaviors was investigated. Alterations in cocaine reward behavior were assessed in the conditioned place preference (CPP) paradigm. Mice were injected with a selective 5-HT2C receptor agonist, Ro 60-0175 (0, 1, 3, 10mg/kg, i.p.) prior to cocaine administration (10mg/kg, i.p.) on cocaine-conditioning days. Administration of Ro 60-0175(10mg/kg) prior to cocaine attenuated the development of cocaine place preference. To assess the potential of the 5-HT2C receptor to influence cocaine-induced behavioral sensitization, mice were pretreated with either saline or Ro 60-0175 (10mg/kg, i.p.) and 30min later, administered cocaine (20mg/kg, i.p.) or saline once daily for 5 days. Locomotor activity was measured daily following cocaine administration. After a 10-day drug-free period, locomotor activity was measured on day 16 following a challenge injection of cocaine (20mg/kg, i.p.). Pharmacological activation of 5-HT2C receptors withRo 60-0175 attenuated acute cocaine-induced activity on days 1–5, as well as the development of long-term cocaine-induced locomotor sensitization. Thus, activation of 5-HT2C receptors attenuated the rewarding and locomotor-stimulating effects of cocaine, as well as inhibited the development of sensitization. The current study shows that 5-HT2C receptor activity exerts an inhibitory influence on the short-term and long-term behavioral responses to cocaine.

Involvement of the 5-HT1A and 5-HT2A/2C serotonin receptors in the anxious behavior of prenatally stressed ovariectomized rats

We compared the effects of an antagonist of 5-HT1A receptors, NAN-190 (0.1 mg/kg intraperitoneally), and an antagonist of 5-HT2A/2C receptors, ketanserin (0.1 mg/kg intraperitoneally), which were chronically injected for 14 days, on the anxiety level in prenatally stressed female rats with an ovariectomyinduced experimental deficiency of estrogens. Chronic administration of ketanserin to ovariectomized prenatally stressed females had an anxiolytic effect and corrected disturbed levels of follitropin, lutropin, and estradiol. Administration of NAN-190 to ovariectomized prenatally stressed rats increased the anxiety level and decreased the ratio between the levels of tropic and peripheral sex hormones.

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Dependencies among Editing Sites in Serotonin 2C Receptor mRNA

The serotonin 2C receptor (5-HT2CR)–a key regulator of diverse neurological processes–exhibits functional variability derived from editing of its pre-mRNA by site-specific adenosine deamination (A-to-I pre-mRNA editing) in five distinct sites. Here we describe a statistical technique that was developed for analysis of the dependencies among the editing states of the five sites. The statistical significance of the observed correlations was estimated by comparing editing patterns in multiple individuals. For both human and rat 5-HT2CR, the editing states of the physically proximal sites A and B were found to be strongly dependent. In contrast, the editing states of sites C and D, which are also physically close, seem not to be directly dependent but instead are linked through the dependencies on sites A and B, respectively. We observed pronounced differences between the editing patterns in humans and rats: in humans site A is the key determinant of the editing state of the other sites, whereas in rats this role belongs to site B. The structure of the dependencies among the editing sites is notably simpler in rats than it is in humans implying more complex regulation of 5-HT2CR editing and, by inference, function in the human brain. Thus, exhaustive statistical analysis of the 5-HT2CR editing patterns indicates that the editing state of sites A and B is the primary determinant of the editing states of the other three sites, and hence the overall editing pattern. Taken together, these findings allow us to propose a mechanistic model of concerted action of ADAR1 and ADAR2 in 5-HT2CR editing. Statistical approach developed here can be applied to other cases of interdependencies among modification sites in RNA and proteins.

Pharmacogenomic Associations With Weight Gain in Olanzapine Treatment of Patients Without Schizophrenia

Pharmacogenomic analyses of weight gain during treatment with second-generation antipsychotics have resulted in a number of associations with variants in ankyrin repeat and kinase domain containing 1 (ANKK1)/dopamine D2 receptor (DRD2) and serotonin 2C receptor (HTR2C) genes. These studies primarily assessed subjects with schizophrenia who had prior antipsychotic exposure that may have influenced the amount of weight gained from subsequent therapies. We assessed the relationships between single-nucleotide polymorphisms (SNPs) in these genes with weight gain during treatment with olanzapine in a predominantly antipsychotic-naive population. The association between 5 ANKK1, 54 DRD2, and 11 HTR2C SNPs and weight change during 8 weeks of olanzapine treatment was assessed in 4 pooled studies of 205 white patients with diagnoses other than schizophrenia who were generally likely to have had limited previous antipsychotic exposure. The A allele of DRD2 rs2440390(A/G) was associated with greater weight gain in the entire study sample (P = .0473). Three HTR2C SNPs in strong linkage disequilibrium, rs6318, rs2497538, and rs1414334, were associated with greater weight gain in women but not in men (P = .0032, .0012, and .0031, respectively). A significant association with weight gain for 2 HTR2C SNPs previously reported associated with weight gain, -759C/T (rs3813929) and -697G/C (rs518147), was not found. Associations between weight gain and HTR2C and DRD2 variants in whites newly exposed to olanzapine may present opportunities for the individualization of medication selection and development based on differences in adverse events observed across genotype groups.

Clozapine-Induced Locomotor Suppression is Mediated by 5-HT2A Receptors in the Forebrain

The need for safer, more effective therapeutics for the treatment of schizophrenia is widely acknowledged. To optimally target novel pharmacotherapies, in addition to establishing the mechanisms responsible for the beneficial effects of antipsychotics, the pathways underlying the most severe side effects must also be elucidated. Here we investigate the role of serotonin 2A (5-HT(2A)), serotonin 2C (5-HT(2C)), and dopamine 2 receptors (D₂) in mediating adverse effects associated with canonical first- and second-generation antipsychotic drugs in mice. Wild-type (WT) and 5-HT(2A) knockout (KO) mice treated with haloperidol, clozapine, and risperidone were assessed for locomotor activity and catalepsy. WT mice showed a marked reduction in locomotor activity following acute administration of haloperidol and high-dose risperidone, which was most likely secondary to the severe catalepsy caused by these compounds. Clozapine also dramatically reduced locomotor activity, but in the absence of catalepsy. Interestingly, 5-HT(2A) KO mice were cataleptic following haloperidol and risperidone, but did not respond to clozapine's locomotor-suppressing effects. Restoration of 5-HT(2A) expression to cortical glutamatergic neurons re-instated the locomotor-suppressing effects of clozapine in the open field. In sum, we confirm that haloperidol and risperidone caused catalepsy in rodents, driven by strong antagonism of D₂. We also demonstrate that clozapine decreases locomotor activity in a 5-HT(2A)-dependent manner, in the absence of catalepsy. Moreover, we show that it is the cortical population of 5-HT(2A) that mediate the locomotor-suppressing effects of clozapine.

Striatal Dopamine Release and Genetic Variation of the Serotonin 2C Receptor in Humans

Mesoaccumbal and nigrostriatal projections are sensitive to stress, and heightened stress sensitivity is thought to confer risk for neuropsychiatric disorders. Serotonin 2C (5-HT(2C)) receptors mediate the inhibitory effects of serotonin on dopaminergic circuitry in experimental animals, and preclinical findings have implicated 5-HT(2C) receptors in motivated behaviors and psychotropic drug mechanisms. In humans, a common missense single-nucleotide change (rs6318, Cys23Ser) in the 5-HT(2C) receptor gene (HTR2C) has been associated with altered activity in vitro and with clinical mood disorders. We hypothesized that dopaminergic circuitry would be more sensitive to stress in humans carrying the Ser23 variant. To test this hypothesis, we studied 54 healthy humans using positron emission tomography and the displaceable D(2)/D(3) receptor radiotracer [(11)C]raclopride. Binding potential (BP(ND)) was quantified before and after a standardized stress challenge consisting of 20 min of moderate deep muscular pain, and reduction in BP(ND) served as an index of dopamine release. The Cys23Ser variant was genotyped on a custom array, and ancestry informative markers were used to control for population stratification. We found greater dopamine release in the nucleus accumbens, caudate nucleus, and putamen among Ser23 carriers, after controlling for sex, age, and ancestry. Genotype accounted for 12% of the variance in dopamine release in the nucleus accumbens. There was no association of Cys23Ser with baseline BP(ND). These findings indicate that a putatively functional HTR2C variant (Ser23) is associated with greater striatal dopamine release during pain in healthy humans. Mesoaccumbal stress sensitivity may mediate the effects of HTR2C variation on risk of neuropsychiatric disorders.

Oligomer Size of the Serotonin 5-Hydroxytryptamine 2C (5-HT2C) Receptor Revealed by Fluorescence Correlation Spectroscopy with Photon Counting Histogram Analysis: EVIDENCE FOR HOMODIMERS WITHOUT MONOMERS OR TETRAMERS

Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) are techniques with single molecule sensitivity that are well suited for examining the biophysical properties of protein complexes in living cells. In the present study, FCS and PCH were applied to determine the diffusion coefficient and oligomeric size of G-protein-coupled receptors. FCS was used to record fluctuations in fluorescence intensity arising from fluorescence-tagged 5-hydroxytryptamine 2C (5-HT(2C)) receptors diffusing within the plasma membrane of HEK293 cells and rat hippocampal neurons. Autocorrelation analysis yielded diffusion coefficients ranging from 0.8 to 1.2 μm(2)/s for fluorescence-tagged receptors. Because the molecular brightness of a fluorescent protein is directly proportional to the number of fluorescent proteins traveling together within a protein complex, it can be used to determine the oligomeric size of the protein complex. FCS and PCH analysis of fluorescence-tagged 5-HT(2C) receptors provided molecular brightness values that were twice that of GFP and YFP monomeric controls, similar to a dimeric GFP control, and unaltered by 5-HT. Bimolecular fluorescence complementation of the N- and C-terminal halves of YFP attached to 5-HT(2C) receptors was observed in endoplasmic reticulum/Golgi and plasma membranes with a brightness equal to monomeric YFP. When GFP-tagged 5-HT(2C) receptors were co-expressed with a large excess of untagged, non-fluorescent 5-HT(2C) receptors, the molecular brightness was reduced by half. PCH analysis of the FCS data were best described by a one-component dimer model without monomers or tetramers. Therefore, it is concluded that 5-HT(2C) receptors freely diffusing within the plasma membrane are dimeric.

Diverse effects of 5-HT2C receptor blocking agents on c-Fos expression in the rat basal ganglia

Serotonin(2C) receptors (5-HT2C) exert continuous control on the activity of specific populations of neurons in the basal ganglia. While antagonists block the effect of endogenous 5-HT at 5-HT2C receptors, the actions of inverse agonists may also involve interruption of activity at constitutively active populations of 5-HT2C receptors. We have evaluated the regional impact of these controls by studying, in rats, the expression of the product of the proto-oncogene c-Fos in rat basal ganglia after peripheral doses of the 5-HT2C antagonist SB 243213 (5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-6-trifluoromethylindoline) and the 5-HT2B/2C inverse agonists SB 206553 (5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole.hydrochloride) and S32006 (N-pyridin-3-yl-1,2-dihydro-3H-benzo[e]indole-3-carboxamide). The results show that 1 and 10mg/kg SB 243213 enhanced equally c-Fos expression in the subthalamic nucleus (STN) and dose-dependently in the striatum and nucleus accumbens core (NAcc). SB 206553 (1–10mg/kg), at 10mg/kg only, enhanced c-Fos expression in STN, striatum (except the dorsomedial part), NAcc, entopeduncular nucleus, substantia nigra pars reticulata (SNr) and compacta (SNc) and ventral tegmental area. S32006 induced a similar increase in c-Fos expression in the medial parts of the striatum and NAcc at doses of 1–10mg/kg while it dose-dependently enhanced c-Fos expression in medial parts of the STN and SNr. None of these drugs induced c-Fos expression in the globus pallidus. The distinct pattern of c-Fos expression elicited by the 5-HT2C antagonist and inverse agonists suggests the existence of cellular and functional heterogeneity in the response of the basal ganglia to drugs inhibiting 5-HT2C receptors.

Agomelatine: a review for general practitioners

AbstractAgomelatine is a novel melatonergic antidepressant that restores disrupted biological rhythms, essentially by resetting the circadian clock. Two different, non-monoaminergic, and possibly synergistic pathways, appear to be involved in its mechanism of action. Agomelatine is a melatonin 1 (MT1) and melatonin 2 (MT2) receptor agonist and serotonin (5-hydroxytryptamine) 2C (5-HT2C) receptor antagonist. It is effective in treating moderate-to-severe depression, alleviating the symptoms of anxiety, and restoring the disrupted sleep patterns that often result from this potentially devastating disease. Agomelatine is generally well tolerated with little, if any, propensity for antidepressant-induced sexual dysfunction, weight gain, or discontinuation reactions. However, it has the potential to cause temporary hepatotoxicity, and liver functions need to be monitored as a result. Clinically, meaningful drug interactions are unlikely, with the exception of co-administered potent cytochrome P 1A2 (CYP1A2) en...

Sa1461 Serotonin 2C Receptor Antagonism is a Potential New Candidate Treatment for Stress-Related Anorexia

Background/Aims: Stress is well known to suppress appetite, ultimately leading to chronic eating disorders that may require medical treatment. However, no treatment exists for stressinduced loss of appetite, because its detailed mechanism remains unknown. Serotonin regulates emotions and food intake, and serotonin 2c receptor (5-HT2cR) is known to control feeding behavior. In this study, we used an urocortin 1 (UCN)-induced stress model to evaluate the effects of central 5-HT2cR and changes in the expression of c-fos mRNA. 5-HT2bR, another serotonin receptor that controls food intake, was also evaluated, and the effects of both receptor antagonists on loss of appetite were compared to investigate the potential of 5-HT2cR antagonist drugs for anorexia treatment. Methods: Intraventricular (ICV) injection of UCN or phosphate-buffered saline was administered to Sprague-Dawley rats, and the brains were collected after perfusion fixation, and fixed whole brains were cut in the coronal plane. The mRNA expression of 5-HT2cR and c-fos was evaluated by in situ hybridization. In addition, double staining of 5-HT2cR was performed for a sample section in which an increased level of c-fos mRNA expression was observed. The selective 5-HT2cR antagonist SB242084, the selective 5-HT2bR antagonist SB215505 (intraperitoneal), or rikkunshito (oral), which shows properties of both 5-HT2cR and 5-HT2bR antagonism, was administered and their respective effects on food intake decreases were evaluated in order to clarify the role of 5-HT2cR activation on stress-induced anorexia. Results: The 5-HT2cR mRNA expression in rats under UCN-induced stress was increased primarily in the solitary tract nucleus (NTS) of the medulla oblongata and in the paraventricular nucleus (PVN) of the hypothalamus. Concurrent enhancement of c-fos mRNA expression was observed. Expression of 5-HT2cR mRNA in the arcuate nucleus was approximately the same in both stress-induced and non-stress-induced groups. Double staining revealed that 5-HT2cR and c-fos were expressed in the same cells in the solitary tract nucleus and PVN. Intraperitoneal administration of 5-HT2cR antagonist SB242084 and oral administration of rikkunshito significantly inhibited food intake decreases in rats exposed to UCN-induced stress; in contrast, administration of the 5-HT2bR antagonist SB215505 did not affect food intake. Conclusion: 5-HT2cR antagonism is a potential candidate for the treatment of stressrelated anorexia. When the corticotrophin-releasing factor (CRF) receptor is activated, stress responses may be enhanced by increased responsiveness of 5-HT2cR on CRF neurons in the PVN. Signals from the vagus nerve may increase responsiveness of 5-HT2cR in the NTS, leading to further negative regulation of food intake.

Sa1462 Obesity in Colorectal Adenomas May Have a Gender Specific Relationship

Background/Aims: Stress is well known to suppress appetite, ultimately leading to chronic eating disorders that may require medical treatment. However, no treatment exists for stressinduced loss of appetite, because its detailed mechanism remains unknown. Serotonin regulates emotions and food intake, and serotonin 2c receptor (5-HT2cR) is known to control feeding behavior. In this study, we used an urocortin 1 (UCN)-induced stress model to evaluate the effects of central 5-HT2cR and changes in the expression of c-fos mRNA. 5-HT2bR, another serotonin receptor that controls food intake, was also evaluated, and the effects of both receptor antagonists on loss of appetite were compared to investigate the potential of 5-HT2cR antagonist drugs for anorexia treatment. Methods: Intraventricular (ICV) injection of UCN or phosphate-buffered saline was administered to Sprague-Dawley rats, and the brains were collected after perfusion fixation, and fixed whole brains were cut in the coronal plane. The mRNA expression of 5-HT2cR and c-fos was evaluated by in situ hybridization. In addition, double staining of 5-HT2cR was performed for a sample section in which an increased level of c-fos mRNA expression was observed. The selective 5-HT2cR antagonist SB242084, the selective 5-HT2bR antagonist SB215505 (intraperitoneal), or rikkunshito (oral), which shows properties of both 5-HT2cR and 5-HT2bR antagonism, was administered and their respective effects on food intake decreases were evaluated in order to clarify the role of 5-HT2cR activation on stress-induced anorexia. Results: The 5-HT2cR mRNA expression in rats under UCN-induced stress was increased primarily in the solitary tract nucleus (NTS) of the medulla oblongata and in the paraventricular nucleus (PVN) of the hypothalamus. Concurrent enhancement of c-fos mRNA expression was observed. Expression of 5-HT2cR mRNA in the arcuate nucleus was approximately the same in both stress-induced and non-stress-induced groups. Double staining revealed that 5-HT2cR and c-fos were expressed in the same cells in the solitary tract nucleus and PVN. Intraperitoneal administration of 5-HT2cR antagonist SB242084 and oral administration of rikkunshito significantly inhibited food intake decreases in rats exposed to UCN-induced stress; in contrast, administration of the 5-HT2bR antagonist SB215505 did not affect food intake. Conclusion: 5-HT2cR antagonism is a potential candidate for the treatment of stressrelated anorexia. When the corticotrophin-releasing factor (CRF) receptor is activated, stress responses may be enhanced by increased responsiveness of 5-HT2cR on CRF neurons in the PVN. Signals from the vagus nerve may increase responsiveness of 5-HT2cR in the NTS, leading to further negative regulation of food intake.

In Vitro Screening for Drug-Induced Depression and/or Suicidal Adverse Effects: A New Toxicogenomic Assay Based on CE-SSCP Analysis of HTR2C mRNA Editing in SH-SY5Y Cells

Many drugs in clinical trials, or already on the market, can have psychiatric side effects, independently of their therapeutic indication (e.g., Acomplia, Taranabant, and Roaccutane). There is currently no in vitro or in vivo approved test for the detection/prediction of such adverse effects, and the Food and Drugs Administration (FDA) can only issue general alerts on specific therapeutic classes. The development of a screening assay is therefore of real interest. The anti-viral and anti-tumor action of human interferon-alpha (hIFNα) is associated with a variety of neuropsychiatric side effects, including major depression, suicidal ideation and suicide. RNA editing of the serotonin 2C receptor (HTR2C) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification, the regulation of which is altered in depressed suicide victims. In this study, we show that in the SH-SY5Y neuroblastoma cell line, hIFNα specifically activates the ADAR1a isoform and thereby modifies the HTR2C mRNA editing profile. As this hIFNα-induced altered profile partly overlaps with that observed in the brain of depressed suicide victims, we investigated whether it could be used as a signature to identify drugs with depression and/or suicidal side effects. By means of the Biocortech proprietary screening assay, which allows the relative quantification of all the edited HTR2C isoforms in a sample, we blind-tested the effect of 50 marketed molecules on HTR2C mRNA editing in SH-SY5Y cells and identified 17 compounds with an IFN-like editing profile. This new toxicogenomic assay can identify compounds with potential psychiatric adverse events with a positive predictive value of 90%.

Middle-aged female rats retain sensitivity to the anorexigenic effect of exogenous estradiol

It is well established that estradiol (E2) decreases food intake and body weight in young female rats. However, it is not clear if female rats retain responsiveness to the anorexigenic effect of E2 during middle age. Because middle-aged females exhibit reduced responsiveness to E2, manifesting as a delayed and attenuated luteinizing hormone surge, it is plausible that middle-aged rats are less responsive to the anorexigenic effect of E2. To test this we monitored food intake in ovariohysterectomized young and middle-aged rats following E2 treatment. E2 decreased food intake and body weight to a similar degree in both young and middle-aged rats. Next, we investigated whether genes that mediate the estrogenic inhibition of food intake are similarly responsive to E2 by measuring gene expression of the anorexigenic genes corticotropin-releasing hormone (CRH), proopiomelanocortin (POMC), the long form of the leptin receptor (Lepr) and serotonin 2C receptors (5HT2CR) and the orexigenic genes agouti-related peptide (AgRP), neuropeptide Y (NPY), prepromelanin-concentrating hormone (pMCH) and orexin in the hypothalamus of young and middle-aged OVX rats treated with E2. As expected, E2 increased expression of all anorexigenic genes while decreasing expression of all orexigenic genes in young rats. Although CRH, 5HT2CR, Lepr, AgRP, NPY and orexin were also sensitive to E2 treatment in middle-aged rats, POMC and pMCH expression were not influenced by E2 in middle-aged rats. These data demonstrate that young and middle-aged rats are similarly sensitive to the anorexigenic effect of E2 and that most, but not all feeding-related genes retain sensitivity to E2.

The N-terminal region of the human 5-HT2C receptor has as a cleavable signal peptide

The 5-hydroxytryptamine 2C (5-HT2C) receptor has a single nucleotide polymorphism (SNP) site at amino acid position 23 in its N-terminal tail. The polymorphism involves conversion of a cysteine to serine. The site, designated C23S, is located within a 32 amino acid long predicted signal peptide. The aim of the present study was to investigate whether the 5-HT2C receptor indeed has a functional cleavable signal peptide. For this purpose, ten N-terminally modified 5-HT2C receptors were constructed. Modifications included addition of the influenza virus hemagglutinin signal peptide, addition of a FLAG epitope, truncation of the N-terminal tail, and combinations of these changes. The receptors were transiently expressed in COS-7 cells. The relative amounts of receptors expressed at the membranes were quantified by [3H]-mesulergine radioligand binding. In one of the receptor constructs the FLAG epitope was inserted just after the endogenous putative signal peptide. Immunostaining with the M1 antibody, which recognizes the FLAG epitope only as free N-terminal entity, was used to detect whether the putative signal peptide preceding the FLAG epitope was cleaved off. The results suggest the following conclusions. The predicted signal peptide in the N-terminal tail of the 5-HT2C receptor acts as a cleavable signal peptide. Cleaving of the signal peptide is important for translocation of the wild type receptor to the plasma membrane. The two amino acids differentially encoded by the C23S SNP are likely absent from the mature 5-HT2C receptor.

Serotonin 2c receptor RNA editing in major depression and suicide

Objectives. mRNA for serotonin 2C receptor (5-HT2CR) undergoes editing which results in numerous isoforms. More highly edited isoforms exhibit decreased function. We recently found greater 5-HT2CR editing in suicide victims with prior bipolar disorder (BPD) or schizophrenia (SZ) compared with non-suicide patients and normal controls (NC). This study compares suicides and non-suicides with major depressive disorder (MDDSuic and MDDNoSuic) and non-suicide NC. Methods. mRNA editing was assessed in prefrontal cortex of 24 MDDSuic, 21 MDDNoSuic, and 56 NC using next generation sequencing. mRNA expression of 5-HT2CR and editing enzymes (ADAR1-2) was assessed by real-time PCR. Results. Editing was lower in MDDNoSuic than in MDDSuic, which did not differ from NC. No differences in the 5-HT2CR or ADAR1 expression were detected. ADAR2 expression was higher in NC than in MDD subjects, but did not differ between MDDNoSuic and MDDSuic. Conclusions. Our findings suggest the presence of two factors associated with 5-HT2CR editing. One factor, which probably stems from decreased ADAR2 expression, is linked to MDD and is associated with less editing. The other, seen also in our previous study of suicide in BP and SZ, is linked to suicide alone and is associated with more editing and, therefore, less receptor function.

Quantitative changes in intracellular calcium and extracellular-regulated kinase activation measured in parallel in CHO cells stably expressing serotonin (5-HT) 5-HT2A or 5-HT2Creceptors

BackgroundThe serotonin (5-HT) 2A and 2C receptors (5-HT2AR and 5-HT2CR) are involved in a wide range of physiological and behavioral processes in the mammalian central and peripheral nervous systems. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. We have developed quantitative assays for cells stably expressing these two receptors involving minimal cell sample manipulations that dramatically improve parallel assessments of two signaling responses: intracellular calcium (Cai++) changes and activation (phosphorylation) of downstream kinases. Such profiles are needed to begin to understand the simultaneous contributions from the multiplicity of signaling cascades likely to be initiated by serotonergic ligands.ResultsWe optimized the Cai++ assay for stable cell lines expressing either 5-HT2AR or 5-HT2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT2AR- and 5-HT2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT2AR-expressing cells showed significant release of Cai++ in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines.ConclusionsThese data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT2AR and 5-HT2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.

A Study of Possible Associations Between Single Nucleotide Polymorphisms in the Serotonin Receptor 1A, 1B, and 2C Genes and Self‐Reported Ejaculation Latency Time

Previous research has indicated that serotonergic genes may influence ejaculatory function. Attempts to investigate effects of polymorphisms in serotonergic genes have been carried out, but so far, no study has conducted exploratory genotype analyses regarding the serotonin receptor 1A, 1B, and 2C subtypes, which have been hypothesized to mediate the inhibitory effects of serotonin on ejaculation in rodents. The aim of the present study was to investigate effects of a total of six single nucleotide polymorphisms (SNPs) located in genes encoding serotonin receptor subtypes 1A, 1B, and 2C on self-reported ejaculation latency time. A retrospective self-report measure of ejaculation latency time was used to investigate ejaculatory function in a population-based sample of 1,399 male twins. DNA was collected using self-administered saliva sampling. Calculations of allelic effects were conducted using the Generalized Estimating Equations module of PASW 18.0, which appropriately controls for between-subjects dependence. Out of six investigated polymorphisms, two SNPs (both serotonin receptor 5-HT(1B) linked) had a significant main effect on ejaculation latency time. Of these, one (rs11568817) remained significant after Bonferroni correction for multiple testing, indicating that individuals homozygous for the G allele had significantly shorter ejaculation latencies. The results of this study support the hypothesis that serotonergic genes play a role in ejaculatory function in the general population. Replication of the results of the present study is warranted.

Effects of Serotonin 2C Receptor Agonists on the Behavioral and Neurochemical Effects of Cocaine in Squirrel Monkeys

Accumulating evidence indicates that the serotonin system modulates the behavioral and neurochemical effects of cocaine, but the receptor subtypes mediating these effects remain unknown. Recent studies have demonstrated that pharmacological activation of the serotonin 2C receptor (5-HT(2C)R) attenuates the behavioral and neurochemical effects of cocaine in rodents, but such compounds have not been systematically evaluated in nonhuman primates. The present experiments sought to determine the impact of pretreatment with the preferential 5-HT(2C)R agonist m-chlorophenylpiperazine (mCPP) and the selective 5-HT(2C)R agonist Ro 60-0175 [(α-S)-6-chloro-5-fluoro-α-methyl-1H-indole-1-ethanamine fumarate] on the behavioral and neurochemical effects of cocaine in squirrel monkeys. In subjects trained to lever-press according to a 300-s fixed-interval schedule of stimulus termination, pretreatment with either 5-HT(2C)R agonist dose-dependently and insurmountably attenuated the behavioral stimulant effects of cocaine. In subjects trained to self-administer cocaine, both compounds dose-dependently and insurmountably attenuated cocaine-induced reinstatement of previously extinguished responding in an antagonist-reversible manner, and the selective agonist Ro 60-0175 also attenuated the reinforcing effects of cocaine during ongoing cocaine self-administration. It is noteworthy that the selective agonist Ro 60-0175 exhibited behavioral specificity because it did not significantly alter nondrug-maintained responding. Finally, in vivo microdialysis studies revealed that pretreatment with Ro 60-0175 caused a reduction of cocaine-induced dopamine increases within the nucleus accumbens, but not the caudate nucleus. These results suggest that 5-HT(2C)R agonists functionally antagonize the behavioral effects of cocaine in nonhuman primates, possibly via a selective modulation of cocaine-induced dopamine increases within the mesolimbic dopamine system and may therefore represent a novel class of pharmacotherapeutics for the treatment of cocaine abuse.

Chronic agomelatine treatment corrects the abnormalities in the circadian rhythm of motor activity and sleep/wake cycle induced by prenatal restraint stress in adult rats

Agomelatine is a novel antidepressant acting as an MT1/MT2 melatonin receptor agonist/5-HT2C serotonin receptor antagonist. Because of its peculiar pharmacological profile, this drug caters the potential to correct the abnormalities of circadian rhythms associated with mood disorders, including abnormalities of the sleep/wake cycle. Here, we examined the effect of chronic agomelatine treatment on sleep architecture and circadian rhythms of motor activity using the rat model of prenatal restraint stress (PRS) as a putative 'aetiological' model of depression. PRS was delivered to the mothers during the last 10 d of pregnancy. The adult progeny ('PRS rats') showed a reduced duration of slow wave sleep, an increased duration of rapid eye movement (REM) sleep, an increased number of REM sleep events and an increase in motor activity before the beginning of the dark phase of the light/dark cycle. In addition, adult PRS rats showed an increased expression of the transcript of the primary response gene, c-Fos, in the hippocampus just prior to the beginning of the dark phase. All these changes were reversed by a chronic oral treatment with agomelatine (2000 ppm in the diet). The effect of agomelatine on sleep was largely attenuated by treatment with the MT1/MT2 melatonin receptor antagonist, S22153, which caused PRS-like sleep disturbances on its own. These data provide the first evidence that agomelatine corrects sleep architecture and restores circadian homeostasis in a preclinical model of depression and supports the value of agomelatine as a novel antidepressant that resynchronizes circadian rhythms under pathological conditions.