Serotonin receptor 2
The possible involvement of the neurotransmitter serotonin (5-HT) and its binding protein (SBP) in cardiac morphogenesis was studied using mouse whole embryo culture. Embryos were cultured before and during the period of endocardial cushion formation [embryonic (E) days 9-12] in the presence of either 5-HT, the monoamine oxidase (MAO) inhibitor nialamide, or an uptake inhibitor (fluoxetine or sertraline). E9 embryos cultured in the presence of 10 microM 5-HT and nialamide exhibit intense 5-HT immunoreactivity (5-HT IR) throughout the myocardium. This staining is greatly diminished by fluoxetine, sertraline, or the absence of nialamide. As morphogenesis proceeds, myocardial staining in embryos exposed to 5-HT becomes restricted to developing endocardial cushion forming regions and is more completely blocked by uptake inhibitors. No evidence for 5-HT synthesis by myocardium is found at any age studied using the precursor L-tryptophan. SBP is present in endocardial cushions in cultured and uncultured embryos. 3H-thymidine autoradiography demonstrates that both fluoxetine and sertraline inhibit proliferation of cardiac mesenchyme, endocardium, and myocardium. These effects are most pronounced when exposure begins at E9 (prior to cushion formation). Dose-dependent effects of 5-HT on migration of outflow tract cells are also observed. Taken together, these results suggest that 5-HT may play a role in cardiac morphogenesis during endocardial cushion formation (Yavarone, 1993).
A specific antiserum directed against the C-terminal portion of the mouse 5-HT2B receptor has been produced and characterized. After affinity purification, this polyclonal antibody recognizes specifically the mouse 5-HT2B receptor. Immunohistochemical analysis of cryosections from various adult mouse tissues reveals a major 5-HT2B receptor expression in stomach, intestine and pulmonary smooth muscles as well as in myocardium. Furthermore, the antiserum recognizes specific areas of the mouse brain, including cerebellar Purkinje cells and their projection areas (Choi, 1996a).
During embryogenesis, serotonin has been reported to be involved in craniofacial and cardiovascular morphogenesis. However, the detailed molecular mechanisms underlying these functions remain unknown. From mouse and human species, 5-HT2B receptors have been cloned that share signal transduction pathways with other 5-HT2 receptor subtypes (5-HT2A and 5-HT2C). In addition to phospholipase C stimulation, it appears that these three subtypes of receptor transduce a common serotonin-induced mitogenic activity, which could be important for cell differentiation and proliferation. The expression of 5-HT2 receptor mRNAs was studied in the mouse embryo. Interestingly, a peak of 5-HT2B receptor mRNA expression is detected 8-9 days postcoitum, whereas there is only low level 5-HT2A and no 5-HT2C receptor mRNA expression at this stage. Expression of this receptor has been confirmed by binding assays using a 5-HT2-specific ligand that revealed a peak of binding to membrane preparations from 9 days postcoitum embryos. In addition, whole mount in situ hybridization and immunohistochemistry on similar stage embryos detect 5-HT2B expression in neural crest cells, heart myocardium and somites. The requirement for functional 5-HT2B receptors between 8 and 9 days postcoitum is supported by culture of embryos exposed to 5-HT2-specific ligands; 5-HT2B high-affinity antagonist such as ritanserin, induces morphological defects in the cephalic region, heart and neural tube. These antagonistic treatments interfere with cranial neural crest cell migration, induce their apoptosis, and are responsible for abnormal sarcomeric organization of the subepicardial layer and for the absence of the trabecular cell layer in the ventricular myocardium. This study is the first report that 5-HT2B receptors are actively mediating the action of serotonin on embryonic morphogenesis, probably by preventing the differentiation of cranial neural crest cells and myocardial precursor cells (Choi, 1997).
A series of experiments was initiated to test the role of 5-HT2B receptor in embryo development using whole embryo culture. Embryos at about the 5 somite-pair stage were incubated for 24 hours in medium containing 1:1 rat serum/Tyrodes salt solution and various serotonergic drugs. Normal rat serum contains a nearly micromolar amount of 5-HT (0.5-1 mM): dialysed serum cannot support embryonic development in the experimental conditions used, even supplemented by 1 mM 5-HT. Therefore, in order to block the action of 5-HT2B receptors, specific antagonists of the 5-HT2 subtypes of receptors (ritanserin, methysergide, mesulergine, ketanserin and mianserin) were selected for their differential affinity for 5-HT2B receptors. Treatment with antagonist induced several reproducible embryonic defects, which were characterized by optical microscopy and by scanning electron microscopy. Ritanserin-treated embryos show a strong growth retardation as compared to controls. The yolk sac circulation is also impaired as indicated by the formation of few blood islands. Moreover, the cephalic region shows apparent defects in flexure, and the forebrain, hindbrain and the first pharyngeal arch develop abnormally. In addition, the epicardial layer of the ventricular wall is swollen. Defects in embryonic turning, in somite number and shape, and in neural tube shape and closure are also frequently observed after ritanserin treatment. Ritanserin induces these defects in all treated embryos at 1 mM concentration, whereas at 100 nM ritanserin, nearly 20% of the embryos are already affected (Choi, 1997).
Defects could be visually detected 6 hours after the beginning of treatment. Simultaneous addition of agonist with ritanserin, 5-HT or the highly specific 5-HT2B agonist N-acetyl-5-HT (NAS), prevents the onset of these defects in almost 50% of the treated embryos. Interestingly, among the other 5-HT antagonists tested, only those having a high affinity for 5-HT2B receptors give a similar phenotype to that of ritanserin. In contrast, the antagonists mianserin and ketanserin, which have a lower affinity for 5-HT2B than for 5-HT2A or 5-HT2C receptors, give a milder phenotype even at a concentration of 10 mM, pointing out a 5-HT2B-mediated action. The defects induced by 5-HT antagonists were further characterized by light and transmission electron microscopy (TEM) on thin sections of these embryos. Toluidine blue stained sections show that ritanserin-treated embryos have hypoplastic pharyngeal arches and an irregular neural tube with dilated blood vessels. It is important to point out that the reduced size of pharyngeal arches is indicative of impaired NC cell migration and/or proliferation. The NC cells in the ritanserin-treated embryos seem not to migrate properly and remain in a more dorsal aspect of the cephalic region than in the control embryos. Although dividing cells can be seen in the epithelium, densely stained pyknotic nuclei, which are indicative of dying cells, are specifically observed in NC cells of the first pharyngeal arch and in the neuroepithelium near the optic stalk. Several characteristics of apoptosis were identified -- cells presenting electron dense nuclei with condensed chromatin; dark cytoplasm, and fragmented cells -- whereas no necrotic cells can be seen after ritanserin treatment. Similar studies of the cardiac region were performed. In ritanserin-treated embryos the heart structure is disorganized. In particular, the bulboventricular groove is not well defined and the swollen atrioventricular canal shows reticulocyte accumulation, indicating an inefficient circulation. The subepicardial layer is thin and the cardiac trabecular cells are absent in the ventricle. However, elongated cells are present in this layer, which normally contains myocardial trabecular stem cells. TEM analysis of thin sections of ritanserin-treated embryos indicates that this compound induces abnormal differentiation of myofilament sarcomeres in the subepicardial layer. These results suggest either a modification of the differentiation program of the myocardial stem cells and/or a deficient migration of the precursors of the trabecular cells (Choi, 1997).
The expression of mRNAs for two 5-HT receptors (5-HT1C, 5-HT2) has been investigated by evaluating in situ hybridization in the prenatal rat CNS. At embryonic day 14 (E14), the highest signal for 5-HT1C is found in the choroid plexus, while the marginal/intermediate (m/i) zones of the midbrain, brain stem (including monoaminergic groups), and spinal cord also display label. By E18-21 a number of more rostral regions contain transcript, including the hippocampus (CA1), in addition to more intense signal in midbrain, brain stem, and spinal cord. Expression in the choroid plexus appears to peak between E16-E18, although considerable hybridization signal remains at E21. 5-HT2 transcripts are also detected at E14. Label is present in m/i zones of the midbrain and in a number of other areas. In comparison to 5-HT1C, 5-HT2 mRNA is distributed over a wider rostral-caudal extent at this age. As with 5-HT1C mRNA, signal increases over rostral and brain stem areas at late gestational ages, with significant labeling appearing in the olfactory bulb, cerebellum, cortical plate and subplate, hippocampus (dentate gyrus), and monoaminergic nuclei. 5-HT1C and 5-HT2 receptor transcripts are also present over the meninges at E16 and may represent transient expression of these receptors. These expression patterns in the embryonic rat brain, in conjunction with previous evidence indicating that 5-HT can act as a differentiation signal for target neurons, suggests that prenatal 5-HT receptors are positioned to play a role in the prenatal development of the CNS (Hellendall, 1993).
An RNase protection assay (RPA) has been developed that allows the simultaneous analysis of 5-HT2AR, 5-HT2BR, and 5-HT2CR in each sample of RNA. This multiprobe set also comprises probes for two house-keeping genes, L32 and GAPDH, which control for sample-loading errors. Using this RPA probe set, the relative expression of 5-HT2AR, 5-HT2BR, and 5-HT2CR has been examined in rat embryos from embryonic day (ED) 9 to 21 (inclusive) of development. 5-HT2AR levels gradually increase from ED11 to ED21. The expression of 5-HT2BR decreases between ED9 to ED11 then remains relatively constant through ED21. 5-HT2CR is initially expressed at residual levels between ED9 and ED12 but dramatically increases to a peak level at ED13, then decreases by ED17. Expression of the 5-HT2 receptors in these tissues is independently confirmed by RT-PCR, which indicates that there is developmental regulation in the expression of these receptors. The 5-HT2R multiprobe assay will be useful for detecting relative changes in the expression of these receptors in developmental, normal and pathological tissues as well as for monitoring relative changes in expression resulting from the use of pharmaceutical agents (Wu, 1999).
Serotonin, via 5-HT2B receptor, is involved in Xenopus retinal histogenesis and eye morphogenesis by supporting cell proliferation and survival. To analyze the 5-HT2B function in retinal development, a loss-of-function study was performed using both a pharmacological and a morpholino antisense oligonucleotide approach. Gain-of-function experiments were made by microinjecting 5-HT2B mRNA. Misregulation of the 5-HT2B receptor activity causes alterations in the proliferation rate and survival of retinal precursors, resulting in abnormal retinal morphology, where lamination is severely compromised. Clones derived from lipofected retinoblasts that overexpress 5-HT2B show an increase in the relative percentage of ganglion cells, possibly due to protection from apoptosis. This effect is reversed in clones lipofected with a 5-HT2B-specific morpholino. It is hypothesized that the survival of the correct number of ganglion cells is controlled by 5-HT/5-HT2B signaling. Serotonin, acting as a neurotrophic factor, may contribute by refining retinal connectivity and cytoarchitecture (De Lucchini, 2005).
In the developing embryonic mouse hindbrain, synchronized spontaneous activity is driven by midline serotonergic neurons at E11.5. This is mediated, at least in part, by the 5-HT2A receptor, which is expressed laterally in the hindbrain. Activity at E11.5 is widespread within the hindbrain tissue, propagating from the midline to more lateral regions. Using rapid acquisition of [Ca2+]i events along the midline, it has been shown that the rostral midline, primarily in the region of former rhombomere r2, is the primary initiating zone for this activity. It is proposed that at E11.5, the combined events along the rostral-caudal axis in combination with events propagating along the medial-lateral axis could assign positional information to developing neurons within the hindbrain. With further development, to E13.5, both the lateral and caudal dimensions of spontaneous activity retract to the rostral midline, occupying an area only 14% of that exhibited at E11.5. Increased levels of [K+]o (to 8 mM) at E13.5 are able to increase the spread of spontaneous activity laterally and rostro-caudally. This suggests that spontaneous activity in the hindbrain depends in a dynamic way on the dominant initiating zone of the rostral midline, and that this relationship changes over development (Hunt, 2006a).
In the developing embryonic mouse hindbrain, widespread synchronized spontaneous activity at E11.5 retracts to the initiating zone of the rostral hindbrain by E13.5, and ceases completely by E14.5. At E11.5 and E13.5, the primary driver of spontaneous activity is serotonergic input, while other transmitters (GABA, glutamate, NE, and ATP) have only modulatory roles. Using immunocytochemistry, it is also shown that at E13.5, 5-HT-positive neurons in the midline extend over a larger rostro-caudal distance than at E11.5, and that in the presumptive initiating zone, cell bodies occupy a band that extends 200 microm laterally on each side of the midline, with extensive axonal processes. The 5-HT2A receptor retains expression in lateral tissue over this developmental time. In addition to being sensitive to 5-HT receptor antagonists, spontaneous activity is also abolished by blockers of gap junctions, and is increased in frequency and lateral spread by application of ammonium, presumably via increased intracellular pH augmenting gap junction conductance. Thus, 5-HT neurons of the midline remain the primary drivers of spontaneous activity at several stages of development in the hindbrain, relying in part on gap junctional communication during initiation of activity (Hunt, 2006b).
Dopamine (DA) transmission is regulated by serotonin-2C (5-HT2C) receptors, but the site(s) in the brain where these receptors are localized is not known. The present work utilized in vivo microdialysis to investigate the modulation of DA release by 5-HT2C receptors localized in the nerve terminal regions of the mesocortical and nigrostriatal DA pathways. Microdialysis probes implanted in the striatum or the prefrontal cortex (PFC) measured dialysate DA concentrations, while the selective 5-HT2B/2C inverse agonist SB 206553 was given locally by reverse dialysis into these terminal regions. Additionally, the effects of the 5-HT2C agonist mCPP on striatal DA were measured. Local administration of SB 206553 into the striatum increased DA efflux in a concentration-dependent manner. Systemic administration of mCPP decreased striatal DA and attenuated the SB 206553-induced increase. In contrast, infusion of SB 206553 by reverse dialysis into the PFC had no significant effect on basal DA efflux in this region. Additionally, high concentrations of SB 206553 had no effect on high potassium (K+)-stimulated DA release in the PFC. These data contribute to a body of evidence indicating that 5-HT2C receptors inhibit nigrostriatal dopaminergic transmission. In addition, the results suggest that the nigrostriatal system is regulated by 5-HT2C receptors localized in the dorsal striatum. Elucidating the mechanisms by which serotonin modulates striatal and prefrontocortical DA concentrations may lead to improvements in the treatment of diverse syndromes such as schizophrenia, Parkinson's disease, anxiety, drug abuse, and/or depression (Alex, 2005).
Studies on 3,4-methylenedioxymethamphetamine ('ecstasy' or MDMA)-induced neurotoxicity mainly focus on damage of serotonergic terminals. Less attention has been given to neuronal cell death produced by MDMA and other amphetamines in areas including the cortex, striatum and thalamus. In the present study MDMA-induced neurotoxicity was studied in neuronal serum free cultures from rat cortex. Since MDMA intake induces hyperthermia in both animals and humans, the experiments were performed under normal (36.5°C) and hyperthermic conditions (40°C). The findings showed a dose-, time- and temperature-dependent apoptotic cell death induced by MDMA in cortical neurons. MDMA-induced damage was potentiated under hyperthermia. The neurotoxicity was reduced by the serotonin 2A-receptor antagonists in both normothermic and hyperthermic conditions. A model agonist for the serotonin 2A-receptor also induced a dose- and time-dependent apoptotic cell death. Again, protection was provided by antagonists against agonist-induced neurotoxicity, thereby indicating that the MDMA stimulation of the serotonin 2A-receptor leads to neurotoxicity. This study provides for the first time evidence that direct MDMA serotonin 2A-receptor stimulation leads to neuronal cortical death. alpha-Phenyl-N-tert-butyl nitrone a free radical scavenger and the nitric oxide synthase inhibitor Nomega-nitro-L-arginine as well as the NMDA-receptor antagonist MK-801 provided protection under normothermia and hyperthermia, thereby suggesting the participation of free radicals in MDMA-induced cell death. Since MDMA serotonin 2A-receptor agonistic properties lead to neuronal death, clinically available atypical antipsychotic drugs with serotonin 2A-antagonistic properties could be a valuable therapeutic tool against MDMA-induced neurodegeneration (Capella, 2006).
The liver can regenerate its volume after major tissue loss. In a mouse model of liver regeneration, thrombocytopenia, or impaired platelet activity resulted in the failure to initiate cellular proliferation in the liver. Platelets are major carriers of serotonin in the blood. In thrombocytopenic mice, a serotonin agonist reconstituted liver proliferation. The expression of 5-HT2A and 2B subtype serotonin receptors in the liver increased after hepatectomy. Antagonists of 5-HT2A and 2B receptors inhibited liver regeneration. Liver regeneration was also blunted in mice lacking tryptophan hydroxylase 1, which is the rate-limiting enzyme for the synthesis of peripheral serotonin. This failure of regeneration was rescued by reloading serotonin-free platelets with a serotonin precursor molecule. These results suggest that platelet-derived serotonin is involved in the initiation of liver regeneration (Lesurtel, 2006)
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