Serotonin receptor 2c-expressing cells in the ventral CA1 control attention via innervation of the Edinger-Westphal nucleus.

The hippocampus is divided into dorsal and ventral zones along its principal axis. The dorsal hippocampus is critical for learning and memory, yet the basic function of the ventral hippocampus remains elusive. Here we genetically manipulate a subset of excitatory neurons expressing the serotonin receptor 2c (Htr2c) in the ventral hippocampus. Genetically modified virus tracing reveals that these Htr2c cells establish monosynaptic excitatory connections with newly identified neurons in the Edinger-Westphal nucleus (EW), which directly innervate the medial prefrontal cortex. Inactivation of Htr2c cells impairs behavioral performance in a visual-detection task that demands attention, without affecting novel-object recognition, learning, or memory. This attention deficit was recapitulated by inhibition of EW cells and rescued by activation of EW cells or synaptic projections from Htr2c cells onto EW cells. This study uncovers a synaptic pathway for control of attention.

The posterior pituitary expresses the serotonin receptor 2C.

The serotonin receptor 2C (5HT2C) is an important drug target to treat obesity and depression. Its pre-mRNA undergoes alternative splicing, encoding a short RNA1 isoform that is localized intracellularly and a full-length isoform (RNA2) that can reach the cell membrane. These splicing isoforms are deregulated in Prader-Willi syndrome (PWS), due to the loss of a trans-acting regulatory RNA, SNORD115. Here we show that the 5HT2C mRNA is expressed in the posterior pituitary, suggesting that 5HT2C mRNA is generated in the hypothalamus and subsequently conveyed by axonal transport. In the pituitary, the ratio of 5HT2C isoforms is regulated by feeding, and can be manipulated using a splice-site changing oligonucleotide injected into the blood. The pituitary expression of the 5HT2C mRNA may constitute a previously unknown mechanism whereby serotonin in the circulation or drugs targeting the 5HT2C might induce side-effects. Finally, the deregulation of 5HT2C splicing isoforms in PWS could contribute to the known hormonal imbalances.

Enhanced Expression of Serotonin Receptor 5-Hydroxytryptamine 2C is Associated with Increased Feather Damage in Dongxiang Blue-Shelled Layers.

The gene encoding the serotonin receptor 5-hydroxytraptamine 2C (HTR2C) has been implicated in behavioral phenotypes in a number of species. In previous studies, a mutation in the chicken HTR2C gene was found to be associated with feather condition, thereby suggesting a relationship between the gene and receiving feather pecking activity. The present study analyzed the chicken HTR2C gene at both the genomic make-up and expression level in Dongxiang blue-shelled layer. A significant association between the single nucleotide polymorphism (SNP) rs13640917 (C/T) and feather condition was confirmed in the Chinese local layer. Enhanced HTR2C gene expression (151.1-fold) that was associated with high feather damage indicated that the right cerebrum might be the critical region for HTR2C to participate in the regulation of receiving feather pecking behavior.

Oligonucleotide-induced alternative splicing of serotonin 2C receptor reduces food intake.

The serotonin 2C receptor regulates food uptake, and its activity is regulated by alternative pre-mRNA splicing. Alternative exon skipping is predicted to generate a truncated receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated receptor sequesters the full-length receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full-length to truncated receptor protein. Decreasing the amount of truncated receptor results in the accumulation of full-length, constitutively active receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the serotonin 2C receptor and increases pro-opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild-type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood-brain barrier and its systemic delivery reduced food intake in wild-type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the serotonin 2C receptor, indicating that therapies aimed to change pre-mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader-Willi syndrome.

Cocaine modulation of frontostriatal expression of Zif268, D2, and 5-HT2c receptors in high and low impulsive rats.

Impulsivity shares high comorbidity with substance abuse in humans, and high impulsivity (HI) in rats has been identified as a predictive factor for cocaine addiction-like behavior. Despite the evidence that high impulsivity is associated with altered function of corticostriatal networks, the specific neural substrates underlying the increased vulnerability of impulsive individuals to develop cocaine addiction remain unknown. We therefore investigated specific neural correlates of HI within the corticostriatal circuitry and determined how they interact with a protracted history of cocaine self-administration. We used in situ hybridization to map brain expression of two major genes implicated in impulsivity, encoding the dopamine D2 receptor (DA D2R) and the 5-HT2c receptor (5-HT2cR), and an immediate early gene associated with neuronal plasticity, zif268, in groups of rats selected for HI and low impulsivity (LI) on a 5-choice serial reaction time task (5-CSRTT) immediately after 5-CSRTT training, and following 10 or 50 days of cocaine self-administration. HI rats exhibited decreased DA D2R mRNA in the mesolimbic pathway, and increased 5-HT2cR mRNA in the orbitofrontal cortex compared with LI rats. HI rats also showed decreased zif268 mRNA in the ventral and dorsomedial striatum. Cocaine exposure decreased striatal D2R mRNA in both HI and LI rats, decreased 5-HT2cR mRNA differentially in striatal and prefrontal areas between HI and LI rats, and selectively decreased zif268 mRNA in the orbitofrontal and infralimbic cortices of HI animals. These findings implicate novel markers underlying the vulnerability of impulsive rats to cocaine addiction that localize to the OFC, infralimbic cortex, and striatum.

The time course of serotonin 2C receptor expression after spinal transection of rats: an immunohistochemical study.

In the spinal cord serotonin (5-HT) systems modulate the spinal network via various 5-HT receptors. Serotonin 2A receptor and serotonin 2C receptor (5-HT2A and 2C receptors) are likely the most important 5-HT receptors for enhancing the motoneuron excitability by facilitating the persistent inward current (PIC), and thus play an important role for the pathogenesis of spasticity after spinal cord injury. In conjunction with our 5-HT2A receptor study, using a same sacral spinal transection rat model we have in this study examined 5-HT2C receptor immunoreactivity (5-HT2CR-IR) changes at seven different time intervals after spinal injury. We found that 5-HT2CR-IR was widely distributed in different regions of the spinal gray matter and was predominantly located in the neuronal somata and their dendrites although it seemed also present in axonal fibers in the superficial dorsal horn. 5-HT2CR-IR in different regions of the spinal gray matter was seen to be increased at 14days after transection (with an average ∼1.3-fold higher than in sham-operated group) but did not reach a significant level until at 21days (∼1.4-fold). The increase sustained thereafter and a plateau level was reached at 45days (∼1.7-fold higher), a value similar as that at 60days. When 5-HT2CR-IR analysis was confined to the ventral horn motoneuron somata (including a proportion of proximal dendrites) a significant increase was not detected until 45days post-operation. 5-HT2CR upregulation in the spinal gray matter is confirmed with Western blot in the rats 60days post-operation. The time course of 5-HT2CR upregulation in the spinal gray matter and motoneurons was positively correlated with the development of tail spasticity (clinical scores). This indicates that 5-HT2CR is probably an important factor underlying this pathophysiological development by increasing the excitability of both motoneurons and interneurons.

Disease-specific expression of the serotonin-receptor 5-HT(2C) in natural killer cells in Alzheimer's dementia.

Alzheimer's dementia (AD) is a degenerative brain disorder characterized mainly by cholinergic failure, but other neuro-transmitters are also deficient especially at late stages of the disease. Misfolded ß-amyloid peptide has been identified as a causative agent, however inflammatory changes also play a pivotal role. Even though the most prominent pathology is seen in the cognitive functions, specific abnormalities of the central nervous system (CNS) are also reflected in the periphery, particularly in the immune responses of the body. The aim of this study was to characterize the dopaminergic and serotonergic systems in AD, which are also markedly disrupted along with the hallmark acetyl-choline dysfunction. Peripheral blood mono-nuclear cells (PBMCs) from demented patients were judged against comparison groups including individuals with late-onset depression (LOD), as well as non-demented and non-depressed subjects. Cellular sub-populations were evaluated by mono-clonal antibodies against various cell surface receptors: CD4/CD8 (T-lymphocytes), CD19 (B-lymphocytes), CD14 (monocytes), and CD56 (natural-killer (NK)-cells). The expressions of dopamine D(3) and D(4), as well as serotonin 5-HT(1A), 5-HT(2A), 5-HT(2B) and 5-HT(2C) were also assessed. There were no significant differences among the study groups with respect to the frequency of the cellular sub-types, however a unique profound increase in 5-HT(2C) receptor exclusively in NK-cells was observed in AD. The disease-specific expression of 5-HT(2C), as well as the NK-cell cyto-toxicity, has been linked with cognitive derangement in dementia. These changes not only corroborate the existence of bi-directional communication between the immune system and the CNS, but also elucidate the role of inflammatory activity in AD pathology, and may serve as potential biomarkers for less invasive and early diagnostic purposes as well.

Cloning and expression of a zebrafish 5-HT(2C) receptor gene.

The 5-HT(2C) receptor is one of 14 different serotonin (5-HT) receptors that control neural function and behavior. Here, we present the entire sequence of a zebrafish 5-HT(2C) receptor cDNA including the 3' untranslated region and the previously unknown 5' untranslated region. The cloned 5-HT(2C) receptor gene is located on chromosome 7, is approximately 202 kbp long, and contains six exons. The coding region of the gene is 1557 bp long and flanked by a 504 bp 5' UTR and a 1474 bp 3' UTR. The deduced protein sequence of 518 amino acids aligns with orthologs of other vertebrates and is 54% identical to the human and mouse 5-HT(2C) receptor protein sequences. The region of the cDNA that encodes the 2nd cytoplasmic loop of the protein shows a 66% identity with vertebrate orthologs and clearly identifies the gene as a 5-HT(2C) receptor gene. Coupling sites for beta-arrestin and calmodulin are conserved in zebrafish. In-situ hybridization shows that the receptor is expressed in the brain and spinal cord including areas such as the olfactory bulb, the dorsal thalamus, the posterior tuberculum, the hypothalamus and the medulla oblongata. Reverse Transcriptase-PCR experiments indicate that the receptor gene can also be active in other tissues such as skin, ovaries, and axial muscle of adult zebrafish. Expression of the 5-HT(2C) receptor during ontogeny was found as early as 2.5 hpf. Five edited adenines in the region of the human, rat and mouse mRNA that encodes the 2nd cytoplasmic loop are conserved in the zebrafish transcript. However, RNA editing was not detected in the zebrafish. The results characterize the zebrafish 5-HT(2C) receptor gene and gene expression pattern for the first time. The similarities to mammalian 5-HT(2C) receptor genes suggest the use of zebrafish for the study of 5-HT(2C) receptor function in behavior, development and drug discovery.

Effects of 5-HT(2A) and 5-HT(2C) receptor antagonists on acute and chronic dyskinetic effects induced by haloperidol in rats.

An important limitation of classical antipsychotic drugs such as haloperidol (HAL) is their liability to induce extrapyramidal motor symptoms acutely and tardive dyskinetic syndromes when given chronically. These effects are less likely to occur with newer antipsychotic drugs, an attribute that is often thought to result from their serotonin-2 (5-HT(2)) receptor antagonistic properties. In the present study, we used selected doses of the 5-HT(2A) antagonist M100,907, the 5-HT(2C) antagonist SB242,084 and the mixed 5-HT(2A/C) antagonist ketanserin to re-examine the respective roles of 2A vs. 2C 5-HT(2) receptor subtypes in both acute and chronic motor effects induced by HAL. Acutely, SB242,084 (0.5 mg/kg) reduced HAL-induced catalepsy, while M100,907 (0.5 mg/kg) and ketanserin (1 mg/kg) were without effect. None of the drugs reduced HAL-induced Fos expression in the striatum or frontal cortex, and M100,907 actually potentiated HAL-induced Fos expression in the n. accumbens. In rats chronically treated with HAL, both ketanserin and SB242,084 attenuated vacuous chewing movements, while M100,907 had no effect. In addition, 5-HT(2C) but not 5-HT(2A) mRNA levels were altered in several brain regions after chronic HAL. These results highlight the importance of 5-HT2(2C) receptors in both acute and chronic motoric side effects of HAL, and suggest that 5-HT(2C) antagonism could be targeted as a key property in the development of new antipsychotic medications.

5-HT2CRs expressed by pro-opiomelanocortin neurons regulate insulin sensitivity in liver.

Mice lacking 5-HT 2C receptors (5-HT(2C)Rs) displayed hepatic insulin resistance, a phenotype normalized by re-expression of 5-HT(2C)Rs only in pro-opiomelanocortin (POMC) neurons. 5-HT(2C)R deficiency also abolished the anti-diabetic effects of meta-chlorophenylpiperazine (a 5-HT(2C)R agonist); these effects were restored when 5-HT(2C)Rs were re-expressed in POMC neurons. Our findings indicate that 5-HT(2C)Rs expressed by POMC neurons are physiologically relevant regulators of insulin sensitivity and glucose homeostasis in the liver.

Serotonin increases ERK1/2 phosphorylation in astrocytes by stimulation of 5-HT2B and 5-HT2C receptors.

We have previously shown that fluoxetine causes ERK(1/2) phosphorylation in cultured mouse astrocytes mediated exclusively by stimulation of 5-HT(2B) receptors (Li et al., 2008b). This raises the question whether this is also the case for serotonin (5-HT) itself. In the present study serotonin was found to induce ERK(1/2) phosphorylation by stimulation of 5-HT(2B) receptors with high affinity (EC(50): 20-30 pM), and by stimulation of 5-HT(2C) receptor with low affinity (EC(50): 1 microM or higher). ERK(1/2) phosphorylation induced by stimulation of either 5-HT(2B) or 5-HT(2C) receptors was mediated by epidermal growth factor (EGF) receptor transactivation (Peng et al., this issue), shown by the inhibitory effect of AG1478, an inhibitor of the EGF receptor tyrosine kinase, and GM6001, an inhibitor of Zn-dependent metalloproteinases, and thus of 5-HT(2B) receptor-mediated EGF receptor agonist release. It is discussed that the high potency of the 5-HT(2B)-mediated effect is consistent with literature data for binding affinity of serotonin to cloned human 5-HT(2B) receptors and with observations of low extracellular concentrations of serotonin in brain, which would allow a demonstrated moderate and modality-dependent increase in specific brain areas to activate 5-HT(2B) receptors. In contrast the relevance of the observed 5-HT(2C) receptors on astrocytes is questioned.

Functional characteristics of serotonin 5-HT2A and 5-HT2C receptors in the brain and the expression of the 5-HT2A and 5-HT2C receptor genes in aggressive and non-aggressive rats.

The functional activity of serotonin 5-HT(2A) and 5-HT(2C) receptors and the expression of the genes encoding them were studied in Norway rats bred for 60 generations for the presence and absence of high levels of stress-evoked aggression to humans. There were no significant differences in the levels of 5-HT(2A) receptor mRNA in the midbrain, frontal cortex, and hippocampus and the extents of head twitching evoked by the 5-HT(2A) agonist DOI in rats with and without genetically determined high levels of aggression. Administration of the selective 5-HT(2C) agonist MK-212 weakened reflex startle in response to an acoustic signal (the acoustic startle response) in non-aggressive animals but had no significant effects on the response in aggressive animals. Increases in the level of 5-HT(2C) receptor mRNA were seen in the frontal cortex and hippocampus in non-aggressive rats as compared with aggressive animals. Increases in the expression of the 5-HT(2C) receptor gene and the functional state of 5-HT(2C) receptors were seen in the brains of non-aggressive rats, without any changes in the 5-HT(2A) receptor mRNA level or receptor sensitivity; this is evidence for the involvement of 5-HT(2C) receptors in the mechanisms inhibiting fear-evoked aggressive behavior.

Serotonin modulates fast-spiking interneuron and synchronous activity in the rat prefrontal cortex through 5-HT1A and 5-HT2A receptors.

Alterations of the serotonergic system in the prefrontal cortex (PFC) are implicated in psychiatric disorders such as schizophrenia and depression. Although abnormal synchronous activity is observed in the PFC of these patients, little is known about the role of serotonin (5-HT) in cortical synchrony. We found that 5-HT, released by electrical stimulation of the dorsal raphe nucleus (DRN) in anesthetized rats, regulates the frequency and the amplitude of slow (<2 Hz) waves in the PFC via 5-HT(2A) receptors (5-HT(2A)Rs). 5-HT also modulates prefrontal gamma (30-80 Hz) rhythms through both 5-HT(1A)Rs and 5-HT(2A)Rs, but not 5-HT(2C)Rs, inducing an overall decrease in the amplitude of gamma oscillations. Because fast-spiking interneurons (FSi) are involved in the generation of gamma waves, we examined serotonergic modulation of FSi activity in vivo. Most FSi are inhibited by serotonin through 5-HT(1A)Rs, while a minority is activated by 5-HT(2A)Rs, and not 5-HT(2C)Rs. In situ hybridization histochemistry confirmed that distinct populations of FSi in the PFC express 5-HT(1A)Rs and 5-HT(2A)Rs, and that the number of FSi expressing 5-HT(2C)Rs is negligible. We conclude that 5-HT exerts a potent control on slow and gamma oscillations in the PFC. On the one hand, it shapes the frequency and amplitude of slow waves through 5-HT(2A)Rs. On the other hand, it finely tunes the amplitude of gamma oscillations through 5-HT(2A)R- and 5-HT(1A)R-expressing FSi, although it primarily downregulates gamma waves via the latter population. These results may provide insight into impaired serotonergic control of network activity in psychiatric illnesses such as schizophrenia and depression.

Mutation screening and assessment of the effect of genetic variations on expression and RNA editing of serotonin receptor 2C in the human brain.

AIM: Serotonin receptor 2C (HTR2C) has been postulated as being involved in the etiology or pathophysiology of mental disorders such as bipolar disorder, major depression and schizophrenia. We previously revealed the altered mRNA expression and RNA editing of HTR2C in the postmortem brains of patients with mental disorders. Here we examined the relationship between genetic variations and expression level or RNA editing level of HTR2C in the human brain. METHODS: We performed mutation screening of the HTR2C gene by sequencing all exons, exon-intron boundaries, and promoter region in the same cohort used for expression and RNA editing studies (n = 58). Using the detected genetic variations, we examined the relationship between genetic variations and expression or RNA editing level. RESULTS AND CONCLUSION: We did not find novel mutations or single nucleotide polymorphisms that were specific to patients. Genotype and haplotype-based analyses revealed that genetic variations of HTR2C did not account for observed altered expression or RNA editing level of HTR2C in the brain.

Bi-directional modulation of bed nucleus of stria terminalis neurons by 5-HT: molecular expression and functional properties of excitatory 5-HT receptor subtypes.

Activation of neurons in the anterolateral bed nucleus of the stria terminalis (BNST(ALG)) plays an important role in mediating the behavioral response to stressful and anxiogenic stimuli. Application of 5-HT elicits complex postsynaptic responses in BNST(ALG) neurons, which includes (1) membrane hyperpolarization (5-HT(Hyp)), (2) hyperpolarization followed by depolarization (5-HT(Hyp-Dep)), (3) depolarization (5-HT(Dep)) or (4) no response (5-HT(NR)). We have shown that the inhibitory response is mediated by activation of postsynaptic 5-HT(1A) receptors. Here, we used a combination of in vitro whole-cell patch-clamp recording and single cell reverse transcriptase polymerase chain reaction (RT-PCR) to determine the pharmacological properties and molecular profile of 5-HT receptor subtypes mediating the excitatory response to 5-HT in BNST(ALG) neurons. We show that the depolarizing component of both the 5-HT(Hyp/Dep) and the 5-HT(Dep) response was mediated by activation of 5-HT(2A), 5-HT(2C) and/or 5-HT(7) receptors. Single cell RT-PCR data revealed that 5-HT(7) receptors (46%) and 5-HT(1A) receptors (41%) are the most prevalent receptor subtypes expressed in BNST(ALG) neurons. Moreover, 5-HT receptor subtypes are differentially expressed in type I-III BNST(ALG) neurons. Hence, 5-HT(2C) receptors are almost exclusively expressed by type III neurons, whereas 5-HT(7) receptors are expressed by type I and II neurons, but not type III neurons. Conversely, 5-HT(2A) receptors are found predominantly in type II neurons. Finally, bi-directional modulation of individual neurons occurs only in type I and II neurons. Significantly the distribution of 5-HT receptor subtypes in BNST(ALG) neurons predicted the observed expression pattern of 5-HT responses determined pharmacologically. Together, these results suggest that 5-HT can differentially modulate the excitability of type I-III neurons, and further suggest that bi-directional modulation of BNST(ALG) neurons occurs primarily through an interplay between 5-HT(1A) and 5-HT(7) receptors. Hence, modulation of 5-HT(7) receptor activity in the BNST(ALG) may offer a novel avenue for the design of anxiolytic medications.

Overexpression of 5-HT2C receptors in forebrain leads to elevated anxiety and hypoactivity.

The 5-HT(2C) receptor has been implicated in mood and eating disorders. In general, it is accepted that 5-HT(2C) receptor agonists increase anxiety behaviours and induce hypophagia. However, pharmacological analysis of the roles of these receptors is hampered by the lack of selective ligands and the complex regulation of receptor isoforms and expression levels. Therefore, the exact role of 5-HT(2C) receptors in mood disorders remain controversial, some suggesting agonists and others suggesting antagonists may be efficacious antidepressants, while there is general agreement that antagonists are beneficial anxiolytics. In order to test the hypothesis that increased 5-HT(2C) receptor expression, and thus increased 5-HT(2C) receptor signalling, is causative in mood disorders, we have undertaken a transgenic approach, directly altering the 5-HT(2C) receptor number in the forebrain and evaluating the consequences on behaviour. Transgenic mice overexpressing 5-HT(2C) receptors under the control of the CaMKIIalpha promoter (C2CR mice) have elevated 5-HT(2C) receptor mRNA levels in cerebral cortex and limbic areas (including the hippocampus and amygdala), but normal levels in the hypothalamus, resulting in > 100% increase in the number of 5-HT(2C) ligand binding sites in the forebrain. The C2CR mice show increased anxiety-like behaviour in the elevated plus-maze, decreased wheel-running behaviour and reduced activity in a novel environment. These behaviours were observed in the C2CR mice without stimulation by exogenous ligands. Our findings support a role for 5-HT(2C) receptor signalling in anxiety disorders. The C2CR mouse model offers a novel and effective approach for studying disorders associated with 5-HT(2C) receptors.

[Functional correlates of the brain 5-HT2A and 5-HT2C serotonin receptors and expression of 5-HT2A AND 5-HT2C genes in aggressive and nonaggressive Norway rats].

Functional activity of the brain 5-HT2A and 5-HT2C serotonin receptors and expression of 5-HT2A and 5-HT2C genes in Norway rats selectively bred for 60 generations for high level and for a lack of fear-induced aggression towards humans were studied. There were no essential differences between aggressive and tame rats in 5-HT2A receptor mRNA level in the midbrain, hippocampus and frontal cortex as well as in the intensity of head-twitch induced by selective 5-HT2A agonist DOI. At the same time, administration of 5-HT2C receptor selective agonist MK-212 reduced the amplitude of acoustic startle response in tame but not in aggressive animals. The increase in 5-HT2C receptor mRNA level in the frontal cortex and hippocampus of nonaggressive rats compared to aggressive animals was shown. The data indicate involvement of the brain 5-HT2C receptor rather than 5-HT2C receptors in the mechanisms of genetically predetermined suppression of fear-induced aggressive behavior.

Developmental changes in serotonin 2C receptor mRNA editing in the rat cerebral cortex and primary cultured cortical neurons.

Serotonin 2C receptor (5-HT2CR) mRNA has been reported to receive editing at 5 nucleotide positions (named sites A-E) which are located inconsecutively on the nucleotide sequence encoding the 2nd intracellular loop of the receptor protein. To clarify the physiological role of 5-HT2CR mRNA editing, we investigated developmental changes in editing frequencies at sites A-E in the rat cerebral cortex and primary cultured cortical neurons. The editing at sites A and B increased in parallel with the rat brain development and reached a plateau of 80-100% frequency at postnatal days 1-3. Although editing frequency at site C was low compared to those detected at other sites except site E during a developmental period, it reached the maximal value of 30% during a first 7-d period after birth and then decreased gradually to the negligible level at PN49. Site D exhibited almost constant susceptibility (about 60%) to editing, while no editing at site E was occurred during rat brain development. Similar changes during development in editing frequencies at these sites were observed in primary cultured cortical cells during the cultivation period. These findings indicated that editing sites A-D on 5-HT2CR mRNA have different susceptibility and that the frequencies at these sites are not always constant during development.

Pharmacological characterization of mitogen-activated protein kinase activation by recombinant human 5-HT2C, 5-HT2A, and 5-HT2B receptors.

The type 2 serotonin (5-HT(2)) receptor subfamily is known to couple to phosphoinositide hydrolysis (PI) and the subsequent mobilization of intracellular Ca(2+), as well as the release of arachidonic acid (AA). Less is known of 5-HT(2)-mediated activation of the mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK1/2) signaling. The present study measured the relative efficacies and potencies of 5-HT agonists to activate ERK2 in non-neuronal cells expressing recombinant human 5-HT(2A), 5-HT(2B), and 5-HT(2C(ISV)) receptors. 5-HT agonists stimulated ERK2 activity via all three 5-HT(2) subtypes. There were no meaningful differences in the potencies or relative efficacies of these agonists to affect ERK2 activity vs. PI accumulation or Ca(2+) mobilization, suggesting that these pathways may be sequentially linked. Indeed, ERK2 activity was very sensitive to PKC inhibition and calcium chelation and insensitive to tyrosine kinase and PI-3-kinase inhibition. 5-HT(2) receptors efficiently couple to MAPK activation via sequential PI hydrolysis, and Ca(2+) mobilization. This profile differs from reports of "agonist-directed trafficking of receptor stimulus" between PI/Ca(2+) and AA pathways activated by 5-HT(2) receptors.

Characterization of contractile 5-hydroxytryptamine receptor subtypes in the in situ autoperfused kidney in the anaesthetized rat.

Using several 5-hydroxytryptamine (5-HT) agonists and antagonists, we attempted to characterize the receptor subtypes involved in the contractile response to 5-HT in the in situ autoperfused rat kidney. An intra-arterial (i.a.) bolus injection of 5-HT (0.00000125 to 0.1 microg/kg) increased renal perfusion pressure in a dose-dependent way but did not affect the systemic blood pressure. The selective 5-HT2 receptor agonist alpha-methyl-5-HT (alpha-methyl-5-hydroxytryptamine) and the non-selective 5-HT2C receptor agonist (1-(3-chlorophenyl)piperazine), m-CPP, caused a local vasoconstrictor effect in the autoperfused rat kidney, whereas BW723C86, a selective 5-HT2B receptor agonist, the 5-HT1 receptor agonist 5-carboxamidotryptamine, 5-CT, and the selective 5-HT3 receptor agonist m-CPBG (1-(m-chlorophenyl)-biguanide) did not modify the renal perfusion pressure. The vasoconstrictor effect elicited by alpha-methyl-5-HT and m-CPP was significantly decreased by ritanserin (a 5-HT2 receptor antagonist), SB 206553 (3,5-Dihydro-5-methyl-N-3pyridinylbenzo[1,2.b:4,5-b']dipyrrole(1H)-carboxamide hydrochloride), a selective 5-HT2B/2C receptor antagonist and enalapril, but was not modified by pretreatment with spiperone (a 5-HT2A receptor antagonist). The results of protein expression analyses allow us to postulate that 5HT-SRC (a 5-HT2C receptor protein) is expressed in renal tissue and differentially expressed in renal artery. Our data suggest also that the serotonergic vasoconstrictor response induced in the in situ autoperfused rat kidney would be mediated by local 5-HT2C receptor activation.