5-HT7 Receptor Expression Research Articles

5-HT 2 Receptors Facilitate JC Polyomavirus Entry

The human JC polyomavirus (JCPyV) causes the rapidly progressing demyelinating disease progressive multifocal leukoencephalopathy (PML). The disease occurs most often in individuals with AIDS but also occurs in individuals receiving immunomodulatory therapies for immune-related diseases such as multiple sclerosis. JCPyV infection of host cells requires the pentasaccharide lactoseries tetrasaccharide c (LSTc) and the serotonin receptor 5-hydroxytryptamine (5-HT) receptor 5-HT2AR. While LSTc is involved in the initial attachment of virus to cells via interactions with VP1, the mechanism by which 5-HT2AR contributes to infection is not clear. To further define the roles of serotonin receptors in infection, HEK293A cells, which are poorly permissive to JCPyV, were transfected with 14 different isoforms of serotonin receptor. Only 5-HT2 receptors were found to support infection by JCPyV. None of the other 11 isoforms of serotonin receptor supported JCPyV infection. Expression of 5-HT2 receptors did not increase binding of JCPyV to cells, but this was not unexpected, given that the cells uniformly expressed the major attachment receptor, LSTc. Infection of these cells remained sensitive to inhibition with soluble LSTc, confirming that LSTc recognition is required for JCPyV infection. Virus internalization into HEK293A cells was significantly and specifically enhanced when 5HT2 receptors were expressed. Taken together, these data confirm that the carbohydrate LSTc is the attachment receptor for JCPyV and that the type 2 serotonin receptors contribute to JCPyV infection by facilitating entry.

Brain RegionSpecific Transcriptomic Markers of Serotonin1A Receptor Agonist Action Mediating Sexual Rejection and Aggression in Female Marmoset Monkeys

IntroductionIn a marmoset model of hypoactive female sexual function, we have shown that repeated administration of the serotonin (5HT)1A agonist R(+)8hydroxy2(diNpropylamino)tetralin (8OHDPAT) inhibits sexual receptivity in female marmoset monkeys and increases aggression toward the male pairmate. AimThe aims of this study are to investigate gene expression changes induced by 8OHDPAT in lasermicrodissected brain areas that regulate female sexual function and to identify genes, functional gene classes, and pathways associated with 8OHDPATmediated inhibition of female sexual receptivity. MethodsGene expression was measured in the medial prefrontal cortex (mPFC), medial preoptic area (mPOA), cornu ammonis1 (CA1) area of the hippocampus (CA1), and dorsal raphé nucleus (DRN) of four 8OHDPATtreated (0.1 mg/kg; daily administration for 16 weeks) and four vehicletreated female marmosets using a marmosetspecific microarray (European Marmoset Microarray [EUMAMA]) and validated by realtime quantitative polymerase chain reaction (RTqPCR). Enriched functional gene classes were determined. In a parallel candidate gene approach, the expression of serotonergic candidate genes, i.e., the 5HT1A, 5HT2A, and 5HT7 receptors and the 5HT transporter (5HTT), was measured by RTqPCR. Main Outcome MeasuresThe main outcome is the differential expression of genes between 8OHDPAT and vehicletreated marmosets. Results8OHDPAT affected the gene classes important to neural development (mPFC, mPOA, and DRN), neurotransmission (mPOA), energy production (mPFC and mPOA), learning and memory (CA1), and intracellular signal transduction (DRN). Oxytocin (OXT) in the mPOA and 5HTT in the DRN were strongly increased by 8OHDPAT. 5HT1A tended to increase in the mPFC, while 5HT7 was decreased in the CA1. ConclusionsBrain regionspecific alterations of gene expression regulating neural circuitries, energy demands, and learning processes are associated with 8OHDPATinduced decrease in female sexual receptivity and increase in pairmate aggression. The role of OXT in the serotonergic regulation of female sexual behavior and partner interactions warrants attention in future studies.

Serotonin and the 5‐HT7 receptor: The link between hepatocytes, IGF‐1 and small intestinal neuroendocrine tumors

Platelet-derived serotonin (5-HT) is involved in liver regeneration. The liver is also the metastatic site for malignant enterochromaffin (EC) cell "carcinoid" (neuroendocrine) neoplasms, the principal cellular source of 5-HT. We hypothesized that 5-HT produced by metastatic EC cells played a role in the hepatic tumor-microenvironment principally via 5-HT₇ receptor-mediated activation of hepatocyte IGF-1 synthesis and secretion. Using isolated rat hepatocytes, we evaluated 5-HT₇ receptor expression (using PCR, sequencing and western blot). ELISA, cell transfection and western blots delineated 5-HT-mediated signaling pathways (pCREB, AKT and ERK). IGF-1 synthesis/secretion was evaluated using QPCR and ELISA. IGF-1 was tested on small intestinal neuroendocrine neoplasm proliferation, while IGF-1 production and 5-HT₇ expression were examined in an in vivo SCID metastasis model. Our results demonstrated evidence for a functional 5-HT₇ receptor. 5-HT activated cAMP/PKA activity, pCREB (130-205%, P < 0.05) and pERK/pAKT (1.2-1.75, P < 0.05). Signaling was reversed by the 5-HT₇ receptor antagonist SB269970. IGF-1 significantly stimulated proliferation of two small intestinal neuroendocrine neoplasm cell lines (EC₅₀: 7-70 pg/mL) and could be reversed by the small molecule inhibitor BMS-754807. IGF-1 and 5-HT were elevated (40-300×) in peri-tumoral hepatic tissue in nude mice, while 5-HT₇ was increased fourfold compared to sham-operated animals. We conclude that hepatocytes express a cAMP-coupled 5-HT₇ receptor, which, at elevated 5-HT concentrations that occur in liver metastases, signals via CREB/AKT and is linked to IGF-1 synthesis and secretion. Because IGF-1 regulates NEN proliferation, identification of a role for 5-HT₇ in the hepatic metastatic tumor microenvironment suggests the potential for novel therapeutic strategies for amine-producing mid-gut tumors.

Peripheral 5-HT7 receptors as a new target for prevention of lung injury and mortality in septic rats

Sepsis is a complex pathophysiological event involving metabolic acidosis, systemic inflammatory response syndrome, tissue damage and multiple organ dysfunction syndrome. Although many new mechanisms are being investigated to enlighten the pathophysiology of sepsis, there is no effective treatment protocol yet. Presence of 5-HT7 receptors in immune tissues prompted us to hypothesize that these receptors have roles in inflammation and sepsis. We investigated the effects of 5-HT7 receptor agonists and antagonists on serum cytokine levels, lung oxidative stress, lung histopathology, nuclear factor κB (NF-κB) positivity and lung 5-HT7 receptor density in cecal ligation and puncture (CLP) induced sepsis model of rats. Agonist administration to septic rats increased survival time; decreased serum cytokine response against CLP; decreased oxidative stress and increased antioxidant system in lungs; decreased the tissue NF-κB immunopositivity, which is high in septic rats; and decreased the sepsis-induced lung injury. In septic rats, as a result of high inflammatory response, 5-HT7 receptor expression in lungs increased significantly and agonist administration, which decreased inflammatory response and related mortality, decreased the 5-HT7 receptor expression.In conclusion, all these data suggest that stimulation of 5-HT7 receptors may be a new therapeutic target for prevention of impaired inflammatory response related lung injury and mortality.

Sensitization of restraint-induced corticosterone secretion after chronic restraint in rats: Involvement of 5-HT7 receptors

Serotonin (5-HT) modulates the hypothalamic-pituitary-adrenal (HPA) axis response to stress. We examined the effect of chronic restraint stress (CRS; 20 min/day) as compared to control (CTRL) conditions for 14 days, on: 1) restraint-induced ACTH and corticosterone (CORT) secretion in rats pretreated with vehicle or SB-656104 (a 5-HT7 receptor antagonist); 2) 5-HT7 receptor-like immunoreactivity (5-HT7-LI) and protein in the hypothalamic paraventricular nucleus (PVN) and adrenal glands (AG); 3) baseline levels of 5-HT and 5-hydroxyindolacetic acid (5-HIAA), and 5-HIAA/5-HT ratio in PVN and AG; and 4) 5-HT-like immunoreactivity (5-HT-LI) in AG and tryptophan hydroxylase (TPH) protein in PVN and AG. On day 15, animals were subdivided into Treatment and No treatment groups. Treatment animals received an i.p. injection of vehicle or SB-656104; No Treatment animals received no injection. Sixty min later, Treatment animals were either decapitated with no further stress (0 min) or submitted to acute restraint (10, 30, 60 or 120 min); hormone serum levels were measured. No Treatment animals were employed for the rest of measurements. CRS decreased body weight gain and increased adrenal weight. In CTRL animals, acute restraint increased ACTH and CORT secretion in a time of restraint-dependent manner; both responses were inhibited by SB-656104. Exposure to CRS abolished ACTH but magnified CORT responses to restraint as compared to CTRL conditions; SB-656104 had no effect on ACTH levels but significantly inhibited sensitized CORT responses. In CTRL animals, 5-HT7-LI was detected in magnocellular and parvocellular subdivisions of PVN and sparsely in adrenal cortex. Exposure to CRS decreased 5-HT7-LI and protein in the PVN, but increased 5-HT7-LI in the adrenal cortex and protein in whole AG. Higher 5-HT and 5-HIAA levels were detected in PVN and AG from CRS animals but 5-HIAA/5-HT ratio increased in AG only. Finally, whereas 5-HT-LI was sparsely observed in the adrenal cortex of CTRL animals, it strongly increased in the adrenal cortex of CRS animals. No TPH protein was detected in AG from both animal groups. Results suggest that CRS promotes endocrine disruption involving decreased ACTH and sensitized CORT responses to acute restraint. This phenomenon may be associated with increased function and expression of 5-HT7 receptors as well as 5-HT turnover in AG.

P.1.022 Role of trace amine-associated receptor 1 (TAAR1) in the modulation of the dopaminergic system and cortico-striatal signalling

The 5-HT6 receptor is mainly expressed in CNS regions involved in cognitive processes and has emerged as a promising target for treating cognitive deficits of schizophrenia, inasmuch as their blockade consistently enhances mnemonic performance in rodents [1]. Paradoxically, little is known about 5-HT6 receptorassociated signaling pathways, an issue we have addressed by a proteomic approach. Previously, we demonstrated that mTOR recruitment by prefrontal 5-HT6 receptors was responsible for perturbed cognition in schizophrenia [2]. Here we show that 5-HT6 receptor interacts with Cyclindependant kinase (Cdk)5, a protein involved in actin cytoskeleton rearrangements, which plays a critical role in neurodevelopmental processes. Expressing 5-HT6 receptors in NG108−15 neuroblastoma cells triggered morphological and functional changes associated with neuronal differentiation. These effects were not further enhanced by treating cells with an agonist and were prevented by SB258585, a specific 5-HT6 antagonist, which also impaired association of 5-HT6 receptor with Cdk5. Moreover, 5-HT6 receptor-elicited neuronal differentiation was dependent on Cdk5 activity and required receptor phosphorylation at 350Ser by Cdk5. Supporting a role of endogenously expressed receptors in neuronal differentiation, neurite outgrowth of primary cultured neurons was reduced by either SB258585 or silencing 5-HT6 receptor expression. Complementing work indicating that 5-HT6 receptors modulate neuronal migration [3], the present data show that 5-HT6 receptors promote neuronal differentiation and reveal a critical role for Cdk5 in this process. These novel insights into molecular substrates underlying neurodevelopmental effects of 5-HT6 receptors are of potential importance to the pathophysiology and treatment of developmental psychiatric diseases like autism-spectrum disorder and schizophrenia.

5-HT3 receptor expression in primary human monocytes

There is evidence that 5-hydroxytryptamine-3 (5-HT3) receptor antagonists may be useful for treatment of inflammatory disorders. Studies from human and animal research showed that 5-HT3 receptor antagonists, particularly tropisetron, exert analgesic and anti-inflammatory activity. We have demonstrated that tropisetron inhibited the release of inflammatory mediators such as tumor necrosis factor (TNF) in primary human monocytes. So far, the underlying mechanisms of these effects have not been investigated in detail. This is especially true for the role of the 5-HT3 receptor subtypes A,B,C,D,E in inflammatory events. Here, we investigated the effects of tropisetron and lipopolysaccharide (LPS) on the expression of 5HT3 receptor A,B,C,D,E mRNA levels by real time PCR and FACS analysis. Our data indicate that the different 5-HT3 receptor subtypes are modulated at its transcriptional and surface expression level by inflammatory conditions and 5-HT3 antagonists such as tropisetron in primary human monocytes. 5-HT3 receptor antagonists are therefore a new and promising therapeutic option. New and more selective – in respect to the 5-HT3 subtypes – 5-HT3R antagonists might be a future perspective in the pharmacological treatment of inflammatory diseases such as rheumatoid arthritis.

Preprotachykinin-A Gene Disruption Attenuates Nociceptive Sensitivity After Opioid Administration and Incision by Peripheral and Spinal Mechanisms in Mice

The preprotachykinin A gene (ppt-A) codes for Substance P (SP), supports nociceptive sensitization, and modulates inflammatory responses after incision. Repeated opioid use produces paradoxical pain sensitization—termed opioid-induced hyperalgesia (OIH) —which can exacerbate pain after incision. Here the contribution of SP to peri-incisional nociceptive sensitization and nociceptive mediator production after opioid treatment was examined utilizing ppt-A knockout (−/−) mice and the neurokinin (NK1) receptor antagonist LY303870. Less mechanical allodynia was observed in ppt-A−/− mice compared to wild types (wt) after morphine treatment both before and after incision. Moreover, LY303870 administered with morphine reduced incisional hyperalgesia in wt mice. Incision after saline or escalating morphine treatment upregulated skin IL-1β, IL-6, G-CSF and MIP-1α levels in ppt-A−/− and wt mice similarly. However, chronic morphine treatment greatly exacerbated increases in skin nerve growth factor levels after incision, an effect entirely dependent upon intact SP signaling. Additionally, SP dependent upregulation of prodynorphin, NMDA1 and NK1 receptor expression in spinal cord was seen after morphine treatment and incision. A similar pattern was seen for 5-HT3 receptor expression in tissue from dorsal root ganglia. Therefore, SP may work at both central and peripheral sites to enhance nociceptive sensitization after morphine treatment and incision. PerspectiveThese studies show that SP signaling modulates enhanced nerve growth factor production and changes in neuronal gene expression seen after incision in mice previously exposed to morphine.

Effect of the 5-HT4 receptor and serotonin transporter on visceral hypersensitivity in rats

Visceral hypersensitivity plays an important role in motor and sensory abnormalities associated with irritable bowel syndrome, but the underlying mechanisms are not fully understood. The present study was designed to evaluate the expression of the 5-HT4 receptor and the serotonin transporter (SERT) as well as their roles in chronic visceral hypersensitivity using a rat model. Neonatal male Sprague-Dawley rats received intracolonic injections of 0.5% acetic acid (0.3-0.5 mL at different times) between postnatal days 8 and 21 to establish an animal model of visceral hypersensitivity. On day 43, the threshold intensity for a visually identifiable contraction of the abdominal wall and body arching were recorded during rectal distention. Histological evaluation and the myeloperoxidase activity assay were performed to determine the severity of inflammation. The 5-HT4 receptor and SERT expression of the ascending colon were monitored using immunohistochemistry and Western blot analyses; the plasma 5-HT levels were measured using an ELISA method. As expected, transient colonic irritation at the neonatal stage led to visceral hypersensitivity, but no mucosal inflammation was later detected during adulthood. Using this model, we found reduced SERT expression (0.298 ± 0.038 vs 0.634 ± 0.200, P < 0.05) and increased 5-HT4 receptor expression (0.308 ± 0.017 vs 0.298 ± 0.021, P < 0.05). Treatment with fluoxetine (10 mg·kg−1·day−1, days 36-42), tegaserod (1 mg·kg−1·day−1, day 43), or the combination of both, reduced visceral hypersensitivity and plasma 5-HT levels. Fluoxetine treatment increased 5-HT4 receptor expression (0.322 ± 0.020 vs 0.308 ± 0.017, P < 0.01) but not SERT expression (0.219 ± 0.039 vs 0.298 ± 0.038, P = 0.654). These results indicate that both the 5-HT4 receptor and SERT play a role in the pathogenesis of visceral hypersensitivity, and its mechanism may be involved in the local 5-HT level.

Experimental colitis alters expression of 5-HT receptors and transient receptor potential vanilloid 1 leading to visceral hypersensitivity in mice

Abnormalities of primary afferent nerve fibers are strongly associated with the visceral hypersensitivity state in inflammatory bowel disease. Hypersensitivity of afferent fibers occurs during inflammation. Therefore, to gain an insight into the alterations to receptors and channels expressed in primary afferent neurons, the current study aimed to investigate the time-dependent dynamic changes in levels of 5-hydroxytryptamine (5-HT)3 receptors, 5-HT4 receptors, transient receptor potential vanilloid type 1 (TRPV1) channels, and 5-HT regulatory factors in dextran sulfate sodium (DSS)-induced colitis model mice. 5-HT signaling molecules were detected by indirect staining with specific antibodies. TRPV1-immunoreactivity was detected by staining with fluorescein-conjugated tyramide amplification. To assess nociception, visceromotor responses (VMRs) to colorectal distension were measured by electromyography of abdominal muscles. Immunohistochemical analysis and VMRs to colorectal distention were measured during induction of DSS colitis (days 4 and 7). Inflammation led to downregulation of serotonin transporter immunoreactivities with concomitant increases in 5-HT and tryptophan hydroxylase-1-positive cell numbers. TRPV1-expressing nerve fibers gradually increased during DSS treatment. Abundant nonneuronal TRPV1-immunopositive cell-like structures were observed on day 7 of DSS treatment but not on day 4. The number of 5-HT3 receptor-expressing nerve fibers in the mucosa was increased on day 7. On the other hand, the number of 5-HT4 receptor-expressing nerve fibers in the mucosa decreased on day 7. We made the novel observation of increased expression of neuronal/nonneuronal TRPV1 channels and 5-HT3 receptors, and decreased expression of 5-HT4 receptors in the mucosa in a DSS-induced colitis model. Visceral hyperalgesia was observed on day 7 but not on day 4. A TRPV1 antagonist and a 5-HT3 receptor antagonist attenuated the visceral hyperalgesia to the control level. The alterations of 5-HT signaling via 5-HT3 receptors and of TRPV1 channels in mucosa may contribute to the visceral hypersensitivity in colitis model mice.

Involvement of 5-HT7 receptors in the pathogenesis of temporal lobe epilepsy

The 5-hydroxytryptamine 7 (5-HT7) receptor is the most recently classified member of the serotonin receptor family. The localization of 5-HT7 receptors and the biological activity of its ligands have suggested that 5-HT7 receptors might be involved in the pathogenesis of epilepsy. In the present study, we investigated the correlation between temporal lobe epilepsy and 5-HT7 receptors using pilocarpine-induced rat models of temporal lobe epilepsy and surgical samples of temporal neocortex from intractable epilepsy patients. An analysis of electroencephalogram (EEG) and behavioral changes before and after the treatment of SB269970 hydrochloride (a selective 5-HT7 receptor antagonist, 10mg/kg, i.p.) and AS19 (a selective 5-HT7 receptor agonist, 10mg/kg, s.c.) demonstrated that in epileptic rats the activation of 5-HT7 receptors could increase the number of seizures, which could be reduced by a 5-HT7 receptor antagonist. Moreover, the expression of 5-HT7 receptors was higher in the epilepsy group compared with the nonepileptic group in both rat and human brain tissues. The present results suggested that 5-HT7 receptors participate in the pathogenesis of temporal lobe epilepsy, and a 5-HT7 receptor antagonist may be used as a therapeutic alternative for temporal lobe epilepsy.

Dynamic 5-HT2C receptor editing in a mouse model of obesity.

The central serotonergic signalling system has been shown to play an important role in appetite control and the regulation of food intake. Serotonin exerts its anorectic effects mainly through the 5-HT1B, 5-HT2C and 5-HT6 receptors and these are therefore receiving increasing attention as principal pharmacotherapeutic targets for the treatment of obesity. The 5-HT2C receptor has the distinctive ability to be modified by posttranscriptional RNA editing on 5 nucleotide positions (A, B, C, D, E), having an overall decreased receptor function. Recently, it has been shown that feeding behaviour and fat mass are altered when the 5-HT2C receptor RNA is fully edited, suggesting a potential role for 5-HT2C editing in obesity. The present studies investigate the expression of serotonin receptors involved in central regulation of food intake, appetite and energy expenditure, with particular focus on the level of 5-HT2C receptor editing. Using a leptin-deficient mouse model of obesity (ob/ob), we show increased hypothalamic 5-HT1A receptor expression as well as increased hippocampal 5-HT1A, 5-HT1B, and 5-HT6 receptor mRNA expression in obese mice compared to lean control mice. An increase in full-length 5-HT2C expression, depending on time of day, as well as differences in 5-HT2C receptor editing were found, independent of changes in total 5-HT2C receptor mRNA expression. This suggests that a dynamic regulation exists of the appetite-suppressing effects of the 5-HT2C receptor in both the hypothalamus and the hippocampus in the ob/ob mice model of obesity. The differential 5-HT1A, 5-HT1B and 5-HT6 receptor expression and altered 5-HT2C receptor editing profile reported here is poised to have important consequences for the development of novel anti-obesity therapies.

5-HT7R/G12Signaling Regulates Neuronal Morphology and Function in an Age-Dependent Manner

The common neurotransmitter serotonin controls different aspects of early neuronal differentiation, although the underlying mechanisms are poorly understood. Here we report that activation of the serotonin 5-HT(7) receptor promotes synaptogenesis and enhances synaptic activity in hippocampal neurons at early postnatal stages. An analysis of Gα(12)-deficient mice reveals a critical role of G(12)-protein for 5-HT(7) receptor-mediated effects in neurons. In organotypic preparations from the hippocampus of juvenile mice, stimulation of 5-HT(7)R/G(12) signaling potentiates formation of dendritic spines, increases neuronal excitability, and modulates synaptic plasticity. In contrast, in older neuronal preparations, morphogenetic and synaptogenic effects of 5-HT(7)/G(12) signaling are abolished. Moreover, inhibition of 5-HT(7) receptor had no effect on synaptic plasticity in hippocampus of adult animals. Expression analysis reveals that the production of 5-HT(7) and Gα(12)-proteins in the hippocampus undergoes strong regulation with a pronounced transient increase during early postnatal stages. Thus, regulated expression of 5-HT(7) receptor and Gα(12)-protein may represent a molecular mechanism by which serotonin specifically modulates formation of initial neuronal networks during early postnatal development.

Serotonin in the Gut: What Does It Do?

Serotonin in the Gut: What Does It Do?

2Bs or not 2Bs: that is the question

2Bs or not 2Bs: that is the question

Blockade of 5-HT7 receptors reduces tactile allodynia in the rat

This study assessed the role of systemic and spinal 5-HT7 receptors on rats submitted to spinal nerve injury. In addition, the 5-HT7 receptors level in dorsal root ganglion and spinal cord was also determined. Tactile allodynia was induced by L5/L6 spinal nerve ligation. Systemic (0.01–10mg/kg) or spinal (0.3–30μg) administration of the selective 5-HT7 receptor antagonist SB-269970 but not vehicle reduced in a dose-dependent manner established tactile allodynia. This effect was maintained for about 6h. SB-269970 was more potent and effective by the spinal administration route than through systemic injection. Spinal nerve ligation reduced expression of 5-HT7 receptors in the ipsilateral but not contralateral dorsal root ganglia. Moreover, 5-HT7 receptor levels were lower in the ipsilateral dorsal spinal cord of neuropathic rats compared to naïve and sham rats. No changes in the receptor levels were observed in the contralateral dorsal spinal cord and in both regions of the ventral spinal cord. Data suggest that spinal 5-HT7 receptors play a pronociceptive role in neuropathic rats. Results also indicate that spinal nerve injury leads to a reduced 5-HT7 receptors level in pain processing-related areas which may result from its nociceptive role in this model. Data suggest that selective 5-HT7 receptor antagonists may function as analgesics in nerve injury pain states.

Colonic submucosal 5-HT3 receptor-mediated somatostatin-dependent secretoinhibitory pathway is suppressed in water-immersion restraint stressed rats

We have demonstrated that the activation of 5-hydroxytryptamine (5-HT) receptor 3 in the submucosal plexus suppresses 5-HT-induced colonic ion secretion by increasing submucosal somatostatin release. A number of psychological and physical stresses have impacts on the intestinal mucosal functions, including secretion and the epithelial barrier. Whether the 5-HT3 receptor-mediated somatostatin-dependent secretoinhibitory pathway in the rat distal colon is involved in the stress process is still unknown. The present study aims to investigate the effect of the water-immersion restraint stress on this inhibitory pathway and its underlying mechanisms. Mucosa/submucosa preparations from the rat distal colon were mounted in the Ussing chambers for the measurement of short-circuit current (ISC). Real-time PCR and western blot were performed to study the expression of the 5-HT3 receptor, 5-HT4 receptor, and somatostatin receptor 2. Radioimmunoassay was used to measure somatostatin release. After 2h of water-immersion restraint stress, the membrane resistance (Rte) of rat mucosa/submucosa preparations was significantly decreased, but the baseline ISC and 5-HT-induced ISC responses were significantly increased. The protein expression of the submucosal 5-HT3 receptors and mucosal somatostatin receptor 2 were down-regulated, and the 5-HT-induced somatostatin release from the mucosa/submucosa preparations was significantly reduced in the stress group. Taken together, these results suggest that the 5-HT3 receptor-mediated somatostatin-dependent secretoinhibitory pathway is suppressed in the water-immersion restraint stressed rats, which may contribute to the acute stress-induced increase in colonic secretion.

Immunohistochemical and biomolecular identification of 5-HT7 receptor in rat vestibular nuclei

The association between migraine and balance disorder morbidities has been a topic of interest for many years, and serotonin (5-HT) receptor is known to be closely related with migraine and also to be associated with vestibular symptoms. However, the mechanism underlying the pathogenesis of migrainous vertigo and its association with 5-HT has not been elucidated. Of the many 5-HT receptors, 5-HT₇ receptor has recently attracted attention in the context of migraine treatment. The purpose of this study was to investigate the localization and expression of 5-HT₇ receptor in the rat vestibular nuclei by immunohistochemical staining and reverse transcriptase-polymerase chain reaction (RT-PCR). The present study might provide additional insight into the role of 5-HT₇ receptor in the pathogenesis of migraine-related vestibular symptoms.

Increased expression of the 5-HT6 receptor by viral mediated gene transfer into posterior but not anterior dorsomedial striatum interferes with acquisition of a discrete action–outcome task

Serotonin plays a role in reinforcement learning; however, it is not known which serotonin receptors mediate these effects. Serotonin 6 (5-HT(6)) receptors are abundant in the striatum, a brain area that is involved in reinforcement learning. We previously found that 5-HT(6) receptors in the dorsomedial striatum (DMS) affect reinforcement learning or consolidation over several days. We use viral-mediated gene transfer to discern the role that 5-HT(6) receptors play in mediating post-synaptic responses in anterior versus posterior DMS. Male Long-Evans rats were used to study learning acquisition during a single session of 100 trials on a fixed interval of 20 seconds. In a discrete action-outcome learning task, rats had 10 seconds to press a lever to induce lever retraction and sucrose pellet delivery. In another group of rats, the task had a lever that was continuously extended but only active every 20 seconds, allowing for repetitive, mostly non-reinforced, lever pressing. Results demonstrate that increased expression of 5-HT(6) receptors in the posterior DMS interferes with earning sucrose pellets in only the former task. We take this to indicate that 5-HT(6) receptor signaling in the posterior DMS interferes with acquisition of discrete action-outcome responding.

Investigations on 5-HT4 Receptor Expression and Effects of Tegaserod on Human Platelet Aggregation In Vitro

A meta-analysis of 29 clinical studies on tegaserod revealed an imbalance of cardiovascular ischemic events in patients treated with drug versus placebo. Because increased platelet activity is known to attribute to cardiovascular events, we examined the presence of serotonin receptor type 4 (5-HT₄) receptors and the effects of tegaserod on in vitro aggregation of human platelets. Blood samples were obtained from 20 healthy volunteers and 20 subjects with irritable bowel syndrome with constipation. Samples of whole blood-citrate mixtures were incubated with different tegaserod concentrations mimicking human Cmax values (10 nM), 3.3 times, and 10 times Cmax for at least 1 hour. Conventional plasma platelet aggregation was induced by adenosine diphosphate, collagen, thrombin receptor activating peptide, epinephrine, and serotonin plus adenosine diphosphate. Gene expression analyses targeting 5-HT₄ and serotonin receptor type 2 receptors were carried out using human platelet RNA. The presence of 5-HT₄ receptor protein was investigated by Western blot analysis using membrane fractions from human platelets. Preincubation with tegaserod resulted in mild but statistically significant increases in platelet aggregation induced by adenosine diphosphate, collagen, epinephrine, or serotonin, mostly at supratherapeutic concentrations of tegaserod. The effects were comparable using thrombocytes obtained from healthy volunteers and patients with irritable bowel syndrome with constipation. Expression analysis revealed that mRNA encoding both 5-HT₄ and serotonin receptor type 2 receptors was present in human platelets. The expression of 5-HT₄ receptor mRNA was approximately eightfold lower than serotonin receptor type 2 receptor mRNA. Results from Western blot analyses examining the presence of 5-HT₄ receptor protein in human platelets were in agreement with the findings of the mRNA expression analysis. In platelets harvested from normal persons and patients with irritable bowel syndrome with constipation and exposed in vitro to tegaserod, we detected small but statistically significant concentration-dependent increases in induced platelet aggregation. The relationship of these in vitro effects to clinical cardiovascular ischemic events is presently unclear. Western blot analysis findings suggest the presence of 5-HT₄ receptor protein on human platelets. Further investigations on the potential role of 5-HT₄ receptors in human platelets may be warranted.