InteractiveFly: GeneBrief

5-hydroxytryptamine (serotonin) receptor 2A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - 5-hydroxytryptamine (serotonin) receptor 2A

Synonyms - Serotonin receptor 2

Cytological map position - 82C4--5

Function - G-protein coupled receptor

Keywords - gastrulation

Symbol - 5-HT2A

FlyBase ID: FBgn0087012

Genetic map position - 3-

Classification - serotonin-receptor. seven-pass transmembrane protein

Cellular location - surface transmembrane



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Huser, A., Eschment, M., Gullu, N., Collins, K. A. N., Bopple, K., Pankevych, L., Rolsing, E. and Thum, A. S. (2017). Anatomy and behavioral function of serotonin receptors in Drosophila melanogaster larvae. PLoS One 12(8): e0181865. PubMed ID: 28777821
Summary:
The biogenic amine serotonin (5-HT) is an important neuroactive molecule in the central nervous system of the majority of animal phyla. 5-HT binds to specific G protein-coupled and ligand-gated ion receptors to regulate particular aspects of animal behavior. In Drosophila, as in many other insects this includes the regulation of locomotion and feeding. Due to its genetic amenability and neuronal simplicity the Drosophila larva has turned into a useful model for studying the anatomical and molecular basis of chemosensory behaviors. This is particularly true for the olfactory system, which is mostly described down to the synaptic level over the first three orders of neuronal information processing. This study focused on the 5-HT receptor system of the Drosophila larva. In a bipartite approach consisting of anatomical and behavioral experiments, the distribution and the implications are described of individual 5-HT receptors on naive and acquired chemosensory behaviors. The data suggest that 5-HT1A, 5-HT1B, and 5-HT7 are dispensable for larval naive olfactory and gustatory choice behaviors as well as for appetitive and aversive associative olfactory learning and memory. In contrast, 5-HT/5-HT2A signaling throughout development, but not as an acute neuronal function, affects associative olfactory learning and memory using high salt concentration as a negative unconditioned stimulus. These findings describe for the first time an involvement of 5-HT signaling in learning and memory in Drosophila larvae. In the longer run these results may uncover developmental, 5-HT dependent principles related to reinforcement processing possibly shared with adult Drosophila and other insects.
BIOLOGICAL OVERVIEW

Serotonin (5-hydroxytryptamine, 5-HT) is a well known monoamine neurotransmitter, mitogen, and hormone that mediates a wide variety of physiological effects, including peripheral and central actions. Because serotonin availability is associated with mood disorders in adult humans, it is somewhat surprising that serotonin and its receptors also play a developmental role in flies and humans. In Drosophila, serotonin synchronizes morphological gastrulation movements (Colas, 1999a); in mammals, serotonin regulates morphogenetic functions of cranial neural crest cells and myocardiac cells (Choi, 1997).

Before examining the role of Drosophila Serotonin receptor 2 in gastrulation, the pathway for serotonin synthesis will be briefly described, since this information is important in evaluating the evidence that Serotonin plays a developmental role in Drosophila. For the biosynthesis of 5-HT in mammals, the rate-limiting step is catalysed by tryptophan hydroxylase (TPH) which hydroxylates tryptophan to generate 5-hydroxytryptophan (5-HTP). Mammalian TPH is a homotetramer that uses as cofactors tetrahydrobiopterin (BH4), iron and molecular oxygen. The physiological concentration of tryptophan is subsaturating for TPH. The active pteridin cofactor is reduced BH4, and it is the enzyme dihydropteridin reductase that catalyses BH4's regeneration by the reduction of the dihydrobiopterin. In Drosophila, as in mammals, GTP cyclohydrolase I (GTP-CH) is the first, rate-limiting enzyme in the biosynthesis of pteridins. In Drosophila, GTP-CH is encoded by a single gene, located at the Punch (Pu) locus (McLean, 1993 and Chen, 1994), so named for the mutant eye colors imparted by pteridin deficiencies. 5-HTP is transformed by the enzyme 5-hydroxytryptophan decarboxylase (identical to Dopa decarboxylase, which also transforms L-DOPA into the catecholamine neurotransmitter Dopamine) into serotonin. The L-alpha-aromatic amino acid decarboxylase AADC is a soluble homodimer enzyme whose activity is determined by the concentration of its substrates, 5-HTP and DOPA. Depletion of 5-HT and dopamine in a Drosophila temperature sensitive mutant lacking DDC, leads to learning abnormalities and to an aberrant pattern of serotonergic neurons (Budnik, 1989). In null alleles there is also embryonic lethality associated with an incomplete sclerotization of the cuticle (Wright, 1987). In early gastrulae, a peak of TPH activity (Colas, 1995) preceded by a peak of TPH mRNA (Neckameyer, 1992) is observed. In addition, a peak of DDC activity with unknown function has been described previously at the same embryonic stage (Konrad, 1987). It is concluded that 5-HT may be synthesised zygotically and that consequently, tryptophan and biopterins should be available (Colas, 1999b and references).

The large variety of 5-HT functions is paralleled by the pharmacological complexity of 5-HT receptors that can be classified into different families depending on their signaling pathways. The family including 5-HT1 and 5-HT5 receptors interacts negatively with adenylyl cyclase; the 5-HT2 receptor family is coupled to the activation of phospholipase C (PLC); the family, including 5-HT4, 5-HT6 and 5-HT7 receptors, activates adenylyl cyclase, whereas the 5-HT3 receptor is a ligand gated ion channel. The 5-HT2B receptors from the mouse (Loric, 1992) and human (Choi, 1994) have been cloned. These receptors are functionally coupled to Gq and to the ras signaling pathway, and can be considered as a ligand dependent oncogene acting on protein kinase C (PKC) and MAP Kinase activation (Launay, 1996). Additionally, the Serotonin receptor 2 in Drosophila (5-HT2), the subject of this essay, is expressed during gastrulation. A study of the pharmacological properties of Drosophila 5-HT2 reveals these properties best correlate with mouse 5-HT2B receptor antagonists and the 5-HT2B agonists and that no significant correlation is seen for the 5-HT2A and 5-HT2C agonists. Although Drosophila 5-HT1 subtypes have already been cloned (Hen, 1992), their sequence and pharmacology appear distinct from 5-HT2 (Colas, 1995 and Colas, 1999a).

Embryos lacking the 5-HT2 locus show abnormal germband extension movements. In embryos bearing a deficiency in the 5-HT2 locus [Df(3R)HTRI], time-lapse video recordings reveal an apparently correct cellularization and stage 6 progression: the cephalic furrow forms and both the mesoderm and the proctodeum start to invaginate. The germband initially extends dorsally in response to the pull from the endoderm primordium on the dorsal side of the embryo. However, in homozygous Df(3R)HTRI embryos (genotyped after recording), the extension movements become rapidly delayed. The rapid phase of germband extension, in 5-HT2 null embryos, occurs but at a clearly reduced speed. Taking the initiation of ventral extension movements as a reference time point, the reduced speed of the extension movements appears clearly in 5-HT2 null embryos at stage 7. After a short phase of contraction that corresponds to mesodermal cell shape changes and invagination (210 min) and to the posterior midgut dorsal shift, the ventral extension movements appear reduced both in the initial forward movements and in all the subsequent backward movements. The pole cells invaginate about 3 min late and are not centered. These observations suggest that for mutant embryos, the dorsal contraction is not relayed properly by intercalation: the movements appear slower, delayed and end prematurely. The resulting effect is desynchronization between germband extension and mesodermal and endodermal invaginations and a premature termination of movements (Colas, 1999a).

Transgenic embryos lacking 5-HT2 binding sites mimic Df(3R)HTRI abnormal germband extension. To assess the specific involvement of 5-HT2 in the Df(3R)HTRI deficiency, the expression of the receptor was abolished using an antisense 5-HT2 cDNA expressed from a heat-shock promoter (Y32 transgenic strain). After establishing the conditions of minimal heat-shock, it was verified that in Y32 embryos, the 5-HT2-specific DOI-binding sites (DOI is a specific ligand for 5-HT2 receptor subtypes) are lost at stage 7. An 8 min heat-shock induction is sufficient to eliminate any 5-HT2-specific binding sites and to trigger specific lethality associated with embryonic defects phenocopying those of embryos lacking the 5-HT2 locus. As in Df(3R)HTRI deficiency, slower germband extension movements, abnormal dorsal pole cells positioning and incomplete ventral closure, were observed. In summary, the similar defects in extension movements strongly suggest that both Df(3R)HTRI and the Y32 transgenic strain have lost the same function: signaling through 5-ht2Dro (Colas, 1999a).

Lack of serotonin synthesis in stage-7 embryos mimics the abnormal germband extension seen in Df(3R)HTRI deficiency embryos. Since the peaks of serotonin and serotonin-specific binding sites precisely coincides with stage 7 when the rapid phase of germband extension begins, documented mutations were sought that could specifically affect this peak of serotonin synthesis in the Drosophila gastrula. This study focused on alleles of the Punch locus, which encodes the GTP-CH enzyme (Reynolds, 1987). This enzyme synthesizes the pteridin cofactor required for aromatic amino acids hydroxylases enzymatic activity, including tryptophan hydroxylase, that catalyses the limiting reaction in serotonin synthesis. One class of allele of the Pu locus, the embryo specific or class V, affects maternal and/or early zygotic GTP-CH activity, suggesting that the resulting lethality is due to a deficit in early pteridin function. In embryos homozygous for the punctual mutation rWE67 in the Punch locus, the pushing force generated by ectoderm convergent extension seems also impaired or lacking. A desynchronization of germband extension from mesoderm and endoderm invaginations is revealed. SEM images similar to those obtained from Df(3R)HTRI embryos were obtained for Punch mutants. These images reveal embryos where pole cells, rather than being centered in the forming proctodeal invagination, are located posteriorly, together with defects in the ventral midline closure and arrested extension (Colas, 1999a).

To further assess the ability of the 5-HT2 receptor to control ectodermal cell movements, the global gain of function effect was examined in transgenic embryos expressing a sense 5-HT2 cDNA ectopically under heat-shock promoter control. Strikingly, even after mild induction (heat shock of 8 min), the ubiquitous expression of the receptor strongly disturbs the ectoderm layer elongation and cuticular organization, both associated with a high level of lethality. Therefore, this effect prevents the performance of any reliable phenotypic rescue experiments. Thus the effect of a more restricted ectopic expression was investigated. In the Kr-UT1 strain, which uses a Kruppel driver to express the Gal4 coupled to a UAS-5-HT2 cDNA, a local overexpression of the 5-HT2 mRNA takes place in the domain of the segmentation gap gene Kruppel. It starts almost synchronously with the 5-HT2 receptor endogenous expression as a large domain (parasegment 5±8) in the region of the weakest endogenous receptor expression (parasegment 8) and includes the mesodermal area. Drivers derived from the pair-rule genes generate expression patterns that appear later (due to the delay in synthesizing the Gal4 protein) and therefore are not useful for these studies. In spite of the low level of lethality displayed by Kr-UT1, time-lapse video observations reveal significant perturbations at the beginning of gastrulation. The first defects appear as an abnormal anterior initiation of the extension movements 7 min before the posterior initiation of movements. The midline ectodermal cells first move forward and only when the movements initiate at the posterior pole of the germband does the backward movement start. The pole cells are positioned ahead of endoderm invagination since dorsal movement seems to initiate 1±2 min before the anterior movements start. The initial delay appears to be compensated by a late increase in the speed of cell movement. Globally, the fact that the movement is not strikingly abnormal in timing or extent may explain the low level of lethality. However, the lethality is enhanced when Kr-UT1 gastrulae are heat-shocked in conditions that do not affect the wild-type control (Colas, 1999a).

The delay between anterior and posterior movements is revealed by scanning electron microscopy in embryos where the mesoderm starts closing anteriorly and pole cells, positioned outside at the front of the endoderm invagination, can also be observed. Later (at stage 8), the ectodermal cells located in the dorsal Kruppel domain do not appear to involute into the normal dorsal transverse folds. Instead this domain seems to form a barrier in such a way as to slow the migrating front of the germband. In conclusion, the local gap-like persistence of 5-HT2 also triggers a global decoordination of germband extension with apparent local inhibition of ectoderm movements (Colas, 1999a).

Changes in ectoderm cohesion have been shown to coincide with the onset of cell intercalation. Also they are accompanied by modifications of cellular apical shape (from round to hexagonal and oblong) due to passive stretching in the direction of the ectodermal extension movements. Ultrastructural observations of wild-type embryos have also revealed that junctions in ectodermal cells become apically concentrated from stage 6 to stage 7. Strikingly, in homozygous Df(3R)HTRI embryos at apparent stage 7 (with respect to cephalic furrow and mesodermal invagination) most ectodermal cells have a round apex presenting only few intercellular connecting structures. This is similar to the ectodermal cells in control stage-6 embryos. Thus, the progression in ectoderm cohesion, which normally occurs between stage 6 and 7, is impaired in 5-HT2 null embryos. At gastrulation stage, E-cadherin constitutes the adhesive part of the adherens junction structures, acting by homophilic and calcium-dependent interactions. Junction clustering in the zonula adherens is dependent upon association of the E-cadherin cytoplasmic tail to the cortical actin cytoskeleton. Given the pivotal role of Armadillo in the regulation of E-cadherin-dependent cell adhesive properties, the fraction of this protein associated with E-cadherin at the membrane was analyzed. Confocal microscopy analysis of heat-fixed Df(3R)HTRI homozygous embryos (at stage 7) reveals that the membrane-associated Armadillo, rather than being located as in normal ectodermal cells at their apex (within a length of less than 2 mm), is distributed along their apical side (over more than 4 mm in length). These results reveal in 5-HT2-null stage-7 embryos a reduction of apical projections and an altered apico-basal localization of junctional structures, presumably due to a delay in their apical concentration (Colas, 1999a).

How can the 5-HT2 receptor affect cell intercalation during germ-band extension? Within the early Drosophila gastrula, maternal and zygotic E-cadherin and catenins are ubiquitously distributed. Modulation of adhesive properties within the time-scale of cell intercalation requires regulatory mechanisms that are faster than can be achieved by transcriptional regulation. In 5-HT2 null embryos, the absence of apical ectodermal projections parallels the lack of an apical concentration of adherens junctions. These observations indicate that 5-HT signaling may control the redistribution of pre-existing junctional elements and imply that it can also generate adhesive constraints. As a consequence, the 5-HT2 striped pattern generates parasegmental adhesive differences necessary for cell intercalation. Modulation of adhesive strength by clustering of spot adherens junctions at the apex in zonula adherens is regulated by phosphorylation of intracellular portions of cadherin and/or beta-catenin. Receptors of the 5-HT2 subfamily signal through the trimeric G protein Gq (Launay, 1996). In tumor cells, adhesion mediated by cadherins can be regulated by receptors coupled to Gq (Williams, 1993). In Drosophila, the link between 5-HT2 and cell movements is, therefore, likely to rely on its control of E-cadherin-cytoskeleton association. Preliminary transmission electron microscopy data confirm the abnormal distribution of adherens junction reported here by confocal microscopy studies (Colas, 1999a and references).

Why does elimination of the segmented 5-HT2 expression lead to a non-segmented phenotype? According to the model proposed by Irvine and Wieschaus (1994), intercalation depends upon the establishment of stripes of pair-rule gene products. When these stripes are widened or eliminated, either by pair-rule mutations, mutations in genes that regulate pair-rule gene expression, or ubiquitous expression of eve, then ectodermal cell intercalation and germband extension is reduced. Although the postulated adhesive molecules remain to be identified, the ability of this model to explain the effects of embryonic patterning mutations on germband extension make it attractive. In its simplest form, this model for cell intercalation requires only two types of adhesive cells, which would be distributed in alternate segments. This model implied that (1) within stripes, relative strengths of adhesion among cells must be equal; (2) cells of different stripes must differ quantitatively in their strength of adhesion, and (3) defects resulting from alterations of the segmented pattern may not necessarily be segmented. Furthermore, it has been reported that within a single tissue, cells expressing an identical cadherin can, on the basis of the expression level of this protein, segregate into distinct populations. The data presented in this study indicate that both the lack and the excess of misexpressed 5-HT2 receptors induce abnormal germband extension accompanied by a change in the subcellular distribution of cellular junctions. By inducing local gap-like ectopic 5-HT2Dro, subtle desynchronized movements restricted to the transgene overexpression domain and loss of ectodermal plasticity are observed in the dorsal region of the embryo. This corresponds to the initial location of the Kruppel domain, a region in which the receptor is normally only weakly expressed. This phenotype is reminiscent of that previously reported for embryos expressing eve ectopically; such embryos also display a reduced germband extension (Irvine, 1994). Combined, these data suggest that the alternate distribution of differentially dosed receptor is required to generate normal germband extension and is consistent with 5-HT signaling as a mechanism generating parasegmental differences in adhesion necessary for cell intercalation. Since the defects do not appear to have a segmented pattern, one has to assume that 5-HT signaling is involved in generating segmental differences in the wild-type Drosophila embryo, although direct evidence for an alternate distribution of cell adhesive structures has not yet been reported. The identification of direct intracellular targets of 5-HT signaling that control cell adhesion should constitute an important contribution to the understanding of how extracellular signals control cell movements (Colas, 1999a).


REGULATION

Transcriptional Regulation

The 5-HT2 mRNA stripes appear in phase with those of cells expressing the pair-rule gene fushi tarazu. The 5-HT2 pattern in ftz mutants displays important modification; a partial loss of stripe restriction, shift of the first stripe to the anterior of the cephalic furrow, and enlargement of the low-expressing middle region to the third stripe. Therefore, 5-HT2 transcription is not strictly dependent of ftz expression. The 5-HT2 pattern is uneffected for mutated loci known not to affect ftz, such as engrailed and odd-skipped, and the mesodermic exclusion persists in snail mutants. Therefore, the 5-HT2 gene is located in the vicinity of ftz within the hiearchy of segmentation genes (Colas, 1995).

Serotonin synthesis and germband extension

Given the gastrulation impairments observed in the 5-HT2 receptor mutant, one might predict that similar phenotypes would be observed in 5-HT synthesis mutants. Various Drosophila mutants in which a lack of 5-HT has been detected at the gastrulation stage have embryonic lethality and display abnormal gastrulation movements associated with specific cuticular defects. After the initial description of the 5-HT2 mRNA expression at the early gastrula stage (Colas, 1995), the genomic locus of the 5-HT2 gene was mapped to 82C-E, and two small overlapping deletions were generated. Df(3R)HTR6 and Df(3R)HTRI are homozygous lethal and only the latter deletes the receptor gene. In homozygous Df(3R)HTRI embryos [but not in Df(3R)HTR6], the pushing force generated by ectodermal cell intercalation is impaired or lacking. This is associated with a desynchronization of germband extension from mesoderm and endoderm invaginations. Extreme desynchronization leads to a complete extension arrest. Although the mesoderm invagination is apparently normal in homozygous deficient embryos, defects in the ventral midline closure are frequently observed (Colas, 1999a). Although the phenotype displayed by the dead embryos is variable, a common characteristic is the original differentiation of their cuticle. In addition to 24% of homozygous balancer embryos, which die as weak first instar larvae, 20% of the individually polymerase chain reaction (PCR)-genotyped homozygous Df(3R)HTRI embryos secrete a cuticle with very few structures suggesting that it results from embryos arrested at an early stage (ghost). In the other homozygous Df(3R)HTRI dead embryos, all segments are present and appear identical in contrast to pair-rule mutants. Such a segment, termed 'double-line', consists of only two rows of thick denticles interspersed by clear spaces. Homozygous Df(3R)HTRI embryos and larvae show, within a range of increasing severity of embryonic defects, the double line phenotype either in normal sized crawling trachealess larvae (26%); in small late-hatching and non-crawling first instar larvae (14%) (petite); in non-hatched embryos similar to petite (32%) or in embryos lacking complete cuticular head structures (8%) (punchy). Furthermore, Df(3R)HTR6, j7E8 or j3A4 transposon insertion homozygous embryos do not display any of these characteristic embryonic abnormalities and die later during larval stages.

Examination of morphologically staged, wild-type gastrulae has revealed that the peaks of 5-HT and of 5-HT2 receptor precisely coincide with stage 7 when the rapid phase of germband extension begins (Colas, 1999a). A search was carried out for documented mutations that could specifically affect this peak of 5-HT synthesis in the Drosophila gastrula. The first focus was placed on alleles of the Punch locus, which encodes the GTP-CH enzyme (Reynolds, 1987). This enzyme synthesises the pteridin cofactor required for the enzymatic activity of aromatic amino acid hydroxylases, including tryptophan hydroxylase (TPH). The embryo-specific class of alleles of the Pu locus affects maternal and/or early zygotic GTP-CH activity. It has been suggested that this lethality is due to a deficit in early pteridin function. Using a capillary electrophoresis technique to evaluate 5-HT content in single class V Punch rWE67 embryos, three populations (in stage-7 embryos) can be distinguished: 25.5% with no detectable 5-HT (less than 5 attomoles); 25.5% with an average of 43,80 attomoles and 49.0% with an average of 22 attomoles, while the control embryos contain 44.5 attomoles. Consistently, three different embryo populations are found by PCR-genotyping after 48 h in the rWE67 progeny: 47% give rise to normal larvae (heterozygous rWE67/CyO) and 24% die at the first-instar larval stage (homozygous balancer CyO/CyO). The cuticle of the third population (29%) (homozygous Punch rWE67/rWE67) is abnormal with a distribution of embryos with all segments having an identical pattern, the double line or ghost phenotype. The complete range of these cuticular phenotypes is present in homozygous 5-HT2 null [Df(3R)HTRI] embryos with the exception of cellularization defects that appear to be specific to maternal functions of biopterins (Chen, 1994). In n homozygous rWE67 embryos, the intercalation of ectoderm cells that drives germband extension fails to occur. There is an associated desynchronization of germband extension from mesoderm and endoderm invaginations (Colas, 1999a). The common cuticular defects observed in the 5HT2 null and in this 5-HT deficient allele of Punch thus constitute an additional phenotypic correlation between the absence of 5-HT2 and the lack of its endogenous ligand 5HT in early gastrulae (Colas, 1999b).

The Df(2R)F36 deficiency uncovers most of the Pu transcribed region (McLean, 1993). Nevertheless, early embryonic lethality is not observed in homozygous F36 embryos; instead they show a larval lethality with unpigmented cuticle but no double lines phenotype (Reynolds, 1987). When assessed for the presence of 5-HT, it appears that 100% of each single tested embryo from heterozygous F36 progeny contain approximately 34 0:37 attomoles of 5-HT. This again supports a link between rWE67 early embryonic lethality and the lack of 5-HT (Colas, 1999b).

Previous molecular characterization of the Punch locus has shown that Pu transcripts and protein are expressed during oogenesis and BH4 levels have been reported to be abnormal in oocytes of a viable class V allele (O'Donnell, 1993). It has been hypothesized that defects in maternal deposition of biopterins may be responsible for early embryonic lethality. To verify this hypothesis a test was carried out for the presence of BH4 in early embryos before zygotic transcription starts. Newly laid embryos from an F36 heterozygous cross contain an average of 25 fmol of BH4/mg of total protein, whereas newly laid embryos from a similar rWE67 cross contain no detectable BH4 and control embryos have a BH4 level averaging 61 fmol of BH4/mg of proteins. The prezygotic BH4 level diminishes with time, suggesting it corresponds to a pool of maternally deposited biopterin which is not stable and that zygotically synthesised GTP-CH is required to maintain this level during subsequent embryonic development. However, a maternally deposited pool of BH4 in F36 embryos appears sufficient to satisfy the early embryonic requirement for biopterins and allows them to reach the larval stage. This result strongly supports the notion that the impairment of 5HT synthesis caused by the absence of maternal BH4 is at the origin of gastrulation defects in rWE67 (Colas, 1999b).

In order to confirm that the absence of a maternal pool of BH4 is directly linked with the lack of 5-HT and consequent gastrulation defects, an investigatation was carried out to see if similar embryonic defects could be observed in other 5-HT synthesis mutants. Mutants in the 5-HT biosynthetic pathway, Df(2R)PblX1 lacking tryptophan hydroxylase (TPH), and Df(2R) TW130 lacking dopa decarboxylase (DDC) were selected. Deficiencies that remove the entire locus were selected in order to eliminate the possibility of the expression of compensatory transcripts. Initially, 5HT levels were tested in single embryos from crosses of heterozygous parents. In crosses from both of these deficiencies, one quarter of all stage-7 embryos lack 5-HT and the rest show 5-HT levels not significantly different from wild type. This confirms that zygotically active enzymes are responsible for the peak of 5-HT synthesis observed at gastrulation. The cuticle of the homozygous dead embryos again showed a distribution of double-line and ghost embryos in Df(2R) PblX1 and in Df(2R) TW130. These data support the notion that double-line cuticular segments are the manifestation of a desynchronized extension and suggest that 5-HT signaling is a mechanism regulating cell intercalation. It ensures that the movements of ectoderm are strictly in phase with those of the other germ layers, a necessity for the reproducibility of development (Colas, 1999b).

Serotonin receptor activity is necessary for olfactory learning and memory in Drosophila

Learning and memory in Drosophila is a complex behavior with many parallels to mammalian learning and memory. Although many neurotransmitters including acetylcholine, dopamine, glutamate, and GABA have been demonstrated to be involved in aversive olfactory learning and memory, the role of serotonin has not been well defined. This study presents evidence of the involvement of individual serotonin receptors in olfactory learning and memory in the fly. A pharmacological approach was followed, utilizing serotonin receptor agonists and antagonists, to demonstrate that all serotonin receptor families present in the fly are necessary for short-term learning and memory. Isobolographic analysis utilizing combinations of drugs revealed functional interactions are occurring between 5-HT1A-like and 5-HT2, and 5-HT2 and 5-HT7 receptor circuits in mediating short-term learning and memory. Examination of long-term memory suggests that 5-HT1A-like receptors are necessary for consolidation and important for recall, 5-HT2 receptors are important for consolidation and recall, and 5-HT7 receptors are involved in all three phases. Importantly, the pharmacological results were validated with genetic experiments, and hypomorph strains for 5-HT2Dro and 5-HT1BDro receptors, as well as knockdown of 5-HT7Dro mRNA, were shown to significantly impair performance in short-term memory. These data highlight the importance of the serotonin system and individual serotonin receptors to influence olfactory learning and memory in the fly, and position the fly as a model system to study the role of serotonin in cognitive processes relevant to mammalian CNS function (Johnson, 2011).

Serotonin 5-HT1A/1BDro, 5-HT2Dro, and 5-HT7Dro receptors are involved in aspects of both short-term and long-term conditioned stimulus olfactory aversive learning and memory processes. One significant finding of this work is that structures extrinsic to the MBs may be involved at some level in olfactory learning and memory, as 5-HT2Dro and 5-HT7Dro receptors are not known to express in the MBs. For each of the receptor drugs tested, there are no disruptive effects on locomotor activity, and this study has shown that there are no confounding effects on olfaction or shock reactivity. Importantly, to validate the pharmacological data, genetic methods were used, and insertion alleles and knockdown of receptor mRNA also were found to produce significant deficits in performance. Together, these genetic data are consistent with the pharmacological data and demonstrate that each serotonin receptor is necessary for aspects of normal olfactory learning and memory in the fly (Johnson, 2011).

For short-term memory (STM), it cannot yet be said which components require serotonin receptors. They may be only necessary for learning, or memory, or they may be involved in both. Future studies will address this issue in more depth. Although there are a number of genetic tools that can be used to examine neuronal and receptor function, pharmacological methods can often provide unique and complementing information. Therefore, this study followed a pharmacological approach that incorporated dose response strategies to examine relative contributions of each receptor to short-term learning and memory, the nature of functional interactions between individual receptor types and circuits, and to begin to dissect out individual roles in specific components of LTM. The results here support the notion of some degree of receptor selectivity for these drugs, as do previous studies examining serotonin receptors in the fly where it has been shown that these drugs can have very different behavioral effects from one another in multiple behaviors. Nevertheless, the exact affinities for and selectivity of these drugs at their intended target receptor remain to be fully validated in Drosophila, and some caution must be exercised when interpreting these data. Interestingly, the data show that both agonists and antagonists at the same receptors disrupt performance. One may predict that if an antagonist is disruptive, then an agonist may be enhancing and vice versa. This is not always the case with GPCR signaling, and there are reports in the literature of both types of ligands at a given receptor producing disruptive effects. Because serotonin primarily plays a modulatory role in the CNS, the receptors likely require a dynamic response to properly regulate learning and memory processes. The homologous mammalian receptors each exhibit constitutive activity, and the fly receptors are predicted to have similar constitutive activity associated with them. If a certain level of basal activity and dynamic response to input levels of serotonin is required for normal performance and homeostasis, then both agonists and antagonists (inverse agonists, as ketanserin, WAY100635, and SB258719) would lead to a reduction of the ability of the receptors to dynamically respond to serotonin, leading to a degradation of performance. For example, similar phenomena are seen with increased and decreased receptor activity in human behaviors. It was also observed that whereas drugs were able to completely disrupt STM performance, genetic methods only produced ~50%-75% reduction in performance. It is likely that with pharmacological methods, it was possible to completely disrupt receptor function as drug levels increase, but for both the hypomorph strains and the RNAi knockdown studies there still exists a population of normal receptors, albeit reduced in expression, that confer some degree of functionality to the circuitry. Another factor may be time of administration. For the STM experiments, drugs were administered for 48 h to reach steady state levels, which may have greater or even different effects than would be evident with acute administration (Johnson, 2011).

With respect to LTM processes, the effects of IC50 concentrations of drug in the food were used to assess the role of the serotonin receptors on acquisition, consolidation, and retrieval. The data suggest that each receptor/circuit has their own unique contribution to LTM, where 5-HT1A-like receptors are critical for consolidation and important for retrieval, 5-HT2 receptors are important for consolidation and retrieval, and 5-HT7 receptors are important for all three components. The possibility remains, however, that the differential results observed on LTM may simply be due to the drugs having differential off-target effects at other GPCRs that are important for LTM in addition to the core response mediated by the individual 5-HT receptors (Johnson, 2011).

Having established the involvement of serotonin receptors in learning and memory, how might they function in this capacity? The 5-HT1A/1BDro receptors are expressed postsynaptically in the MBs. Localization of these receptors to the MBs strongly implies that they directly influence MB function. Significantly, the 5-HT1A/1BDro receptors are coupled to Gαi and inhibition of adenylate cyclase activity, and when stimulated lead to a reduction of cAMP levels. Levels of cAMP have been demonstrated to be extremely important for learning and memory (Johnson, 2011).

As in mammals, the 5-HT1A-like receptors are also expressed presynaptically and are predicted to have autoreceptor function. The data indicate that administration of the 5-HT1A receptor agonist U92016A in combination with the 5-HT1A receptor antagonist WAY100635 work synergistically to attenuate STM function in the fly. Although these agents are both targeting 5-HT1A receptors, binding affinity and/or functional selectivity at pre- vs. postsynaptic receptors could be promoting a superadditive rather than a subadditive relationship between these two agents. For example, the agonist U92016A may have greater affinity or efficacy at presynaptic receptors and reduce 5-HT release through autoreceptor activity, and the antagonist WAY100635 may have greater affinity or efficacy at postsynaptic receptors to block reception of the signal that together produce a superadditive decrease in 5-HT effects (Johnson, 2011).

A similar synergistic behavioral effect was observed in previous studies with combinations of agonists and antagonists for 5-HT1A-like receptors with respect to aggressive behaviors. Different affinities and efficacies for drugs acting at the same G-protein coupled receptor located at different biological sites is a well-established phenomenon termed functional selectivity, and this pre/postsynaptic phenomenon plays a significant role in the action of drugs, such as aripiprazole, in humans (Johnson, 2011).

5-HT2Dro receptors are located postsynaptically on neurons of the protocerebrum that are in close proximity to Kenyon cells of the MBs. These cells may normally serve to influence the function of Kenyon cells, and 5-HT2Dro receptor activity may therefore conceivably be indirectly modulating function of the MBs. These receptors are coupled to Gαq, and are generally stimulatory in nature and in mammalian CNS are involved in cognitive processing and integrating sensory information. Their role in the fly may be similar and facilitating the integration of sensory information into the MBs. Interaction data show that simultaneous administration of 5-HT2 receptor agonist and antagonist are interfering, which would be predicted for a receptor with only postsynaptic localization. Significantly, 5-HT1A antagonists in combination with 5-HT2 antagonists are interfering, indicating a functional interaction between the two receptor circuitries. In mammals, 5-HT1A and 5-HT2 receptors often functionally antagonize one another, and it may be that blockade of 5-HT2Dro receptors counteracts the effects of blockade of 5-HT1ADro receptors in the fly. In addition, there is expression of 5-HT2Dro in a subset of cells of the EB that may be contributing to its role in learning and memory. This notion is supported by data indicating functional interactions occurring between 5-HT2Dro and 5-HT7Dro receptor circuitry, which has high expression in the EB (Johnson, 2011).

The results examining the 5-HT7Dro receptor are very intriguing. Although the receptor is expressed weakly in other circuits and areas of the brain (Becnel, 2011), its strong expression in all large field R-neurons of the EB is suggestive of involvement of the EB at some level in olfactory learning and memory processes. Previous attempts to study the role of this structure in olfactory learning and memory have largely been unsuccessful. Walking and flying are mediated by the EB, and the use temperature sensitive off/on shibireTS or TRPM channels to inactivate the entire structure, or mutants that structurally disrupt the EB, has been shown to produce profound coordination and locomotor difficulties, precluding accurate testing of the EB’s role in behaviors. An attempt at a more precise analysis examined NMDA receptor function in a subset of EB neurons, and a role has been proposed for consolidation in LTM. Significantly, a subset of large field R-neurons has recently been demonstrated to be necessary for visual pattern memory. Because there are no direct connections between the central complex and the MB, it remains to be elucidated how structures of the central complex are involved in modulating both olfactory and visual memory. Furthermore, the precise 5-HT7Dro expressing neurons extrinsic to the MBs, either within the central complex or elsewhere, modulating learning and memory remain to be determined in future studies (Johnson, 2011).

In summary, this study has provided the first evidence that serotonin receptors are necessary for normal olfactory learning and memory in the fly. STM is disrupted by both pharmacological agents and by genetic manipulations of serotonin receptor function. The use of pharmacological tools has allowed examination of receptor-receptor and receptor-circuitry interactions through isobolographic analysis, where it was determined that there are functional interactions between 5-HT1A and 5-HT2 circuitries, as well as 5-HT2 and 5-HT7 receptor circuitries. These interactions may be interpreted in a model such that 5-HT1A-like receptors expressed within the MBs directly influence MB function for STM, and particularly consolidation in LTM, by virtue of their location in MB neurons. The 5-HT2Dro expressing multipolar neurons in close proximity to the Kenyon cells of the protocerebrum, and neurons within the EB, may then be modulating the activity of the MBs in STM, and in consolidation and retrieval for LTM. The 5-HT7Dro circuitry may be indirectly influencing MB function through modulation of 5-HT2Dro circuits, or potentially other yet to be identified circuits. In this model, components of the central complex like the EB may be playing a master regulatory role for complex behaviors like STM and all three aspects of LTM, rather than a more specific and direct role in olfactory learning and memory per se. This is consistent with our observations that 5-HT7Dro receptor activity is required for other complex behaviors like normal courtship and mating (Becnel, 2011). This work is intended to be presented as an initial characterization of serotonin receptor involvement in olfactory learning and memory; however, additional work remains to fully elucidate the role of serotonin and its receptors in these processes (Johnson, 2011).


DEVELOPMENTAL BIOLOGY

Embryonic

The 5-HT2 mRNA is first detected by in situ hybridization on whole-mount embryos at the beginning of the cellular blastoderm stage (stage 6; 2 hr 50 min of development). This detection revealed seven evenly spaced transverse stripes along the anteroposterior axis of the embryo, a pattern similar to that of the pair-rule genes. Syncytial expression is not detectable, but the striped pattern appears before cephalic furrow formation, at the onset of cellularization. It is restricted to the ectodermal layer of the blastoderm embryo in four-cell-wide stripes, with uneven intensity among each parasegment. The seven stripes persist during germ-band extension and then the expression disappears. The 5-HT2 mRNA stripes are in phase with those of cells expressing the pair-rule gene fushi tarazu. The colocalization of the earliest-appearing stripe of en-expressing cells with the anterior margin of the most anterior 5-HT2 stripe locates the second parasegment and therefore confirms the 5-HT2 phasing with ftz in the even-numbered parasegments. Concomitant with the peak of 5-HT2 mRNA expression, peak amounts of specific 5-HT2 receptor binding sites and ligand are detected in blastoderm embryos. 5-HT2 mRNA reappears in the embryo ventral nerve cord as well as in the larval CNS in a pair of cells per neuromere, starting at stage 13 (Colas, 1995).

Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster

Neuromodulation confers flexibility to anatomically-restricted neural networks so that animals are able to properly respond to complex internal and external demands. However, determining the mechanisms underlying neuromodulation is challenging without knowledge of the functional class and spatial organization of neurons that express individual neuromodulatory receptors. This study describes the number and functional identities of neurons in the antennal lobe of Drosophila melanogaster that express each of the receptors for one such neuromodulator, serotonin (5-HT). Although 5-HT enhances odor-evoked responses of antennal lobe projection neurons (PNs) and local interneurons (LNs), the receptor basis for this enhancement is unknown. Endogenous reporters of transcription and translation for each of the five 5-HT receptors (5-HTRs) were used to identify neurons, based on cell class and transmitter content, that express each receptor. Specific receptor types are expressed by distinct combinations of functional neuronal classes. For instance, the excitatory PNs express the excitatory 5-HTRs (5-HT2 type and 5-HT7), the 5-HT1 type receptors are generally inhibitory, and distinct classes of LNs each express different 5-HTRs. This study therefore provides a detailed atlas of 5-HT receptor expression within a well-characterized neural network, and enables future dissection of the role of serotonergic modulation of olfactory processing (Sizemore, 2016).


EFFECTS OF MUTATION

Embryos lacking the 5-HT2 locus show abnormal germband extension movements. In embryos bearing a deficiency in the 5-HT2 locus [Df(3R)HTRI], time-lapse video recordings reveal an apparently correct cellularisation and stage 6 progression: The cephalic furrow forms and both the mesoderm and the proctodeum start to invaginate. The germband initially extends dorsally in response to the pull from the endoderm primordium on the dorsal side of the embryo. However, in homozygous Df(3R)HTRI embryos (genotyped after recording), the extension movements become rapidly delayed. The rapid phase of germband extension, in 5-HT2 null embryos, occurs but at a clearly reduced speed. In the control embryos, cells at the anterior part of the germband, initially move both ventrally and anteriorly, pushing against the head region, which, by resisting, induces a bending of the cephalic furrow in the ventral region before disappearing. In contrast, in the deficient embryos the cephalic furrow remains roughly straight and visible for most of the rapid phase of germband extension suggesting that the pushing force is impaired or lacking. This absence of a pushing force is also evident later when extension is almost completely abolished in homozygous embryos. Using image analysis of the digital video recording, the speed of cell movements, near the ventral and dorsal midline of the embryo, was assessed. This was made possible by using newly developed software that allows the 'peeling off' of a superficial layer of the embryos in the video images, laying the embryo flat, and accumulating these images over time. This allows the quantification of the speed and extent of individual cell movement at the surface of an embryo and the evaluation of the synchronization of these movements (Colas, 1999a).

Taking the initiation of ventral extension movements as a reference time point, the reduced speed of the extension movements appears clearly in 5-HT2 null embryos at stage 7. After a short phase of contraction that corresponds to mesodermal cell shape changes and invagination (210 min) and to the posterior midgut dorsal shift, the ventral extension movements appear reduced both in the initial forward movements and in all the subsequent backward movements. Only the endoderm invagination movement occurs at approximately normal speed (10mm/min vs. 15 mm/min). The ectodermal cells move at a speed close to that observed after 20 min of germband extension in control embryos (about 4±5 mm/min). The pole cells invaginate about 3 min late and are not centered. These observations suggest that for mutant embryos, the dorsal contraction is not relayed properly by intercalation, the movements appearing slower, delayed and ending prematurely. The resulting effect is desynchronisation between germband extension and mesodermal and endodermal invaginations and a premature termination of movements (Colas, 1999a).

Signs of desynchronization can be visualized by scanning electron microscopy (SEM). In stage-7 embryos, the pole cells, rather than being centered in the forming proctodeal invagination, are located posteriorly, suggesting an impairment of support cell movements dependent on the ectoderm. Extreme desynchronization leading to a complete morphogenetic block is illustrated in older homozygous embryos. Here, elongated amnioserosa cells (typical of stage 8±9) fill an abnormally large dorsal gap between the front of the germband and the cephalic furrow, whereas pole cells are still visible outside and posterior to the internalised proctodeal invagination. Although in Df(3R)HTRI homozygous deficient embryos the mesoderm invagination is apparently normal, defects in the ventral midline closure are frequently observed (Colas, 1999a).


EVOLUTIONARY HOMOLOGS

Drosophila serotonin receptors stimulate fluid secretion in the Malpighian tubules

Every living cell must detect, and respond appropriately to, external signals. Thus, the functions of intracellular second messengers, such as guanosine 3'5'-cyclic monophosphate (cGMP), adenosine 3'5'-cyclic monophosphate (cAMP), and intracellular calcium, are intensively studied. However, artifact-free manipulation of these messengers is problematic, and simple pharmacology may not allow selective intervention in distinct cell types in a real, complex tissue. A method has been devised by which second messenger levels can be manipulated in cells of choice using the GAL4/UAS system. By placing different receptors (rat atrial natriuretic peptide [ANP] receptor and Drosophila serotonin receptors [5HTDro7 and 5HTDro1A]) under UAS control, they can be targeted to arbitrary defined populations of cells in any tissue of the fly, and second messenger levels can be manipulated simply by adding the natural ligand. The potential of the system is illustrated in the Drosophila renal (Malpighian) tubule, where each receptor has been shown to stimulate fluid secretion, to act through its cognate second messenger, and to be blocked by appropriate pharmacological antagonists. The results have uncovered a new role for cGMP signaling in tubules and also demonstrate the utility of the tubule as a possible in vivo test bed for novel receptors, ligands, or agonists/antagonists (Kerr, 2004).

Ectopic expression of the rat ANP receptor (GC-A) in tubules was achieved under control of both principal cell and stellate cell GAL4 drivers and a heat-shock (hs) promoter. Expression of the GC-A transgene was confirmed by RT-PCR. Expression of GC-A in tubules confers sensitivity to ANP, with resultant production of cGMP. Measurement of cAMP levels in tubules that express GC-A in principal cells (using the c42 GAL4 driver), stellate cells (using the c742 driver), or ubiquitously, show that cGMP levels are stimulated neither by GC-A receptor, nor by ANP, alone. ANP raises cGMP in a dose-dependent manner in c42-GC-A and c724-GC-A tubules, with a nearly 4-fold maximal increase in cGMP levels in c42-GC-A tubules and a 2-fold cGMP increase in c724-GC-A tubules. Since stellate cells make up only a small fraction of the tubule volume, the apparently lower fold increase in stellate cell cGMP probably reflects a larger absolute rise in cGMP in these cells. The EC50 for ANP in both principal and stellate cells is similar, 10-8 M (Kerr, 2004).

Similar increases in fluid transport are observed upon stimulation with ANP, when GC-A is expressed either in only principal cells or in stellate cells. Although cGMP signaling has been shown in principal cells, a diuretic role for cGMP in stellate cells had not been demonstrated before. The effects of cGMP in principal and stellate cells are additive; when GC-A is expressed ubiquitously under hs control, maximal secretion rates are higher than when it is targeted to either cell alone (Kerr, 2004).

cGMP signals in principal cells may activate CNG-type calcium channels, resulting in calcium increase and fluid transport. Targeted expression of the calcium reporter, aequorin, was used to measure changes in intracellular calcium ([Ca2+]i) in GC-A tubules. In tubule principal cells, a biphasic elevation of [Ca2+]i is observed upon ANP stimulation, followed by a sustained secondary rise in [Ca2+]i. By contrast, no change in stellate cell [Ca2+]i was observed upon ANP challenge. Thus, major cellular targets for cGMP in principal cells are CNG channels. Stellate cells, however, must contain uncharacterized cGMP-activated targets that modulate fluid transport (Kerr, 2004).

ANP-mediated cGMP signaling and increased fluid transport are a result of specific ligand-receptor interactions, since the ANP antagonist, anantin, abolishes ANP stimulation of both cGMP and fluid transport in hs-GC-A tubules in a dose-dependent manner (Kerr, 2004).

Pilot experiments have shown that Drosophila tubules are insensitive to 5HT; there is thus scope to modulate second messengers by ectopic expression of the cognate GPCRs. The Drosophila 5HT7Dro receptor raises cAMP levels in cultured cells by activating adenylate cyclase. When 5HT7Dro is expressed in either principal or stellate cells using the appropriate GAL4 drivers, 5HT induces dose-dependent production of cAMP. EC50 for 5HT in both lines was 10-7 M. Similarly, the heat shock construct elicits elevation of [cAMP] upon 5HT treatment. Controls (non-heat-shocked hs-5HT7Dro and heat-shocked wild-type or parental lines) show no response to 5HT. Furthermore, there is no detectable impact on cGMP levels when 5HT7Dro is driven either in principal cells, stellate cells, or ubiquitously (Kerr, 2004).

As would be expected from the literature, increased [cAMP] in principal cells stimulates fluid transport. However, as for cGMP, a previously undocumented diuretic role of cAMP in stellate cells was uncovered, and the maximal rates of 5HT-induced fluid secretion observed with c42-5HT7Dro and c724-5HT7Dro tubules appear to be additive: the sum of maximal rates is approximately equal to the maximal 5HT-induced rate observed in heat-shocked hs-5HT7Dro tubules. It was thus hypothesized that, if stellate cells have the machinery to respond functionally to cAMP and cGMP, then they are also likely to have the machinery to produce the signals (Kerr, 2004).

As with GC-A, the possibility that [Ca2+]i may be altered upon activation of 5HT7Dro was investigated. In principal cells of 5HT7Dro/c42-aeq tubules, 5HT stimulation results in a biphasic elevation of [Ca2+]i, similar to that seen in GC-A/c42-aeq tubules. However, no [Ca2+]i rise was observed in 5HT-stimulated 5HT7Dro/c710-aeq tubules. These results are consistent with both cAMP and cGMP acting on a CNG channel that is expressed only in principal, and not in stellate, cells (Kerr, 2004).

It proved possible to reproduce the known pharmacology of this 5HT receptor. The antagonist (+)-butaclamol almost completely attenuated 5HT-stimulated production of cAMP in a dose-dependent manner, with an IC50 of 2.5 × 10-8 M. This agrees precisely with values obtained for the receptor in cell lines. A maximal dose of (+)-butaclamol (10-5 M) also reduced 5HT-stimulated fluid transport in hs-5HT7Dro tubules (Kerr, 2004).

Another Drosophila 5HT receptor, 5HT1ADro, is known to mobilize intracellular calcium. Expression of 5HT1ADro in either principal or stellate cells results in 5HT-induced calcium responses. As would be expected from the actions of capa and leucokinin (ligands known to act through [Ca2+]i), 5HT also stimulated fluid transport, when 5HT1ADro was expressed in either principal cells or stellate cells, and no effect was seen without a GAL4 driver. Although a comprehensive dose-response curve was not performed, 5HT-stimulated fluid transport was inhibited by yohimbine. The effective concentration for yohimbine in tubules (10-5 M) is comparable with the Ki (18 μM) obtained for this receptor in cell lines (Kerr, 2004).

The 5HT1ADro receptor has been previously shown to inhibit adenylate cyclase in vitro. Accordingly, cAMP and cGMP were measured in 5HT-stimulated c42-5HT1ADro and c724-5HT1ADro tubules. Activation of the 5HT1ADro receptor in tubules does not affect cyclic nucleotide levels, and thus the stimulatory effects of 5HT on fluid transport are solely due to stimulation of increased [Ca2+]i (Kerr, 2004).

Although receptor guanylate cyclases (like GC-A) are self-sufficient, the strategy of modulating second messengers by ectopic expression of GPCRs depends on the expression of cognate G proteins in the target cell type. A priori, it was expected that most G proteins would be widely expressed, but this was confirmed in tubules by RT-PCR with primers against all known G protein α, β, and γ subunits. As predicted, all the Gα subunit genes found in Drosophila are expressed in tubules. Tubules also express the full complement of genes encoding the Gγ subunit; the only G protein subunit that does not appear to be expressed in tubules is Gβ76C (Kerr, 2004).

Although a particular target cell type might not express cognate G proteins, an RT-PCR strategy compared with measurement of second messengers should be informative. As well as the apoaequorin calcium reporter, others are now available, and successful cAMP and cGMP measurements in the tiny (160-cell) tubule suggest that radioimmunoassay is sufficiently sensitive for most Drosophila tissues of interest (Kerr, 2004).

Obviously, the target cell must not normally express receptors for, or respond to, the ligand of choice. In the case of Drosophila, this condition is satisfied; there is no atrial natriuretic peptide-like sequence encoded within the Drosophila genome. For 5HT receptors or other GPCRs, more caution must be exercised; nonetheless, standard experimental controls would quickly identify any problems. In the case of 5HTDro1A, expression has only been documented in some cells of the embryonic nervous system; 5HTDro7 expression has been found in cells of the embryonic ventral midline, and in adult head, but not body. It is thus likely that these constructs will be useful in most Drosophila nonnervous tissues (Kerr, 2004).

This study demonstrates successful ectopic expression of vertebrate and Drosophila receptors in Malpighian tubules and quantifies the effects of such expression on signal transduction pathways and physiological output. There are clear results from this approach: (1) the interactions between 3 second messenger pathways were studied in unprecedented detail, in two cell types, in an organotypic context; (2) the results validated all that was known about signaling through known neuropeptides in the tubule; (3) a diuretic role for cAMP and cGMP in stellate cells was demonstrated, inviting the intriguing question as to which extracellular ligands normally activate these pathways; (4) this conserved pharmacology of a vertebrate receptor (rat GC-A) expressed in a model organism illustrates another possibility for functional genomics, i.e., that novel genes could be characterized relatively cheaply and easily by ectopic expression in a model organism where detailed organotypic-phenotypic analysis is possible. The Drosophila Malpighian tubule is an ideal such 'test bed' for genes where organotypic analysis may be important for normal function (Kerr, 2004).

The utility of this experimental system is more general, allowing sensitive and specific intervention in second messenger signaling in any Drosophila tissue for which there exists a GAL4 driver. Combined with the ready availability of real-time calcium reporters in Drosophila and the possibility of measuring cAMP and cGMP similarly, using transgenic FRET reporters, this simple model organism now has an impressive genetic toolbox for cell signaling studies (Kerr, 2004).

Cloning and characterization of 5-HT2B receptors

Serotonin [5-hydroxytryptamine (5-HT)] modulates feeding activity, egg-laying, and mating behavior in the free-living nematode, Caenorhabditis elegans. A novel receptor cDNA has been cloned from C. elegans (5-HT2Ce) that has high sequence homology with 5-HT2 receptors from other species. When transiently expressed in COS-7 cells, 5-HT2Ce exhibits 5-HT binding activity and activates Ca2+-mediated signaling in a manner analogous to other 5-HT2 receptors. However, 5-HT2Ce displays unusual pharmacological properties, which resemble both 5-HT2 and 5-HT1-like receptors but do not correlate well with any of the known 5-HT2 subtypes. Two splice variants of 5-HT2Ce that differ by 48 N-terminal amino acids were identified. The two isoforms have virtually identical binding and signaling properties but differ in their levels of mRNA expression, with the longer variant being four times more abundant than the shorter species in all developmental stages tested. Taken together, these results describe two variants of a novel C. elegans 5-HT receptor, which has some of the properties of the 5-HT2 family but whose pharmacological profile does not conform to any known class of receptor (Hamdan, 1999).

A G-protein-coupled receptor (5-HT2Lym) resembling members of the 5-HT2 receptor subfamily was cloned from the mollusc Lymnaea stagnalis. Serotonin induces a concentration-dependent increase in intracellular inositol phosphates in HEK293 cells expressing this receptor. 5-HT2Lym differs from mammalian 5-HT2 receptors by the presence of a large amino-terminal region. This large domain appears to preclude an adequate level of expression of 5-HT2Lym in HEK293. Therefore, a cDNA encoding an amino-terminally truncated receptor (delta N-5-HT2Lym) was constructed that appears to be much better expressed in HEK293 cells. delta N-5-HT2Lym-expressing cells exhibit a serotonin-induced stimulation of phosphatidylinositol bisphosphate hydrolysis and a high-affinity binding of the 5-HT2-selective antagonist [3H]mesulergine. Inhibition of this binding by several 5-HT2 antagonists and agonists reveals a pharmacological profile most closely resembling those of 5HT2Dro, 5-HT2B and 5-HT2C (Gerhardt, 1996).

The novel serotonin receptor 5-HT2B shows the highest homology to the 5-HT2 family of receptors. The pharmacological profile of membranes from 5-HT2B cDNA stably transfected LMTK- cell line, corresponds to a new 5-HT2-like receptor named 5-HT2B, although some differences exist between the mouse and rat pharmacology. A similar pharmacological profile is detected on the immortalized teratocarcinoma-derived cell line 1C11 upon 2 days of serotoninergic differenciation by cAMP. In both cell lines, the analysis 125I-DOI binding reveals the presence of a single class of sites, the affinity of which is one order of magnitude lower than the one reported for the 5-HT2A receptor. This demonstrates that the 5-HT2B receptor is functionally expressed before the complete serotoninergic differentiation of 1C11 cells. These observations are in good agreement with the presence of 5-HT2B mRNA in early mouse embryonic development. Furthermore, the major sites of 5-HT2B mRNA embryonic expression are in the heart and in the neural fold, before the closure of the neural tube. Therefore, this receptor could account, at least in part, for the trophic functions attributed to the 5-HT2-like receptors (Choi, 1994a).

The human serotonin 5-HT2B receptor, isolated from a human liver cDNA library, was transfected in COS-1 cells. Its pharmacological profile shows divergence with serotonin 5-HT2B receptors of other species. In particular, although strong correlation is observed between the human and the rat 5-HT2B receptor pharmacology, the correlation is almost as significant for the mouse 5-HT2B and the human 5-HT1D receptor agonists. The major sites of expression of its mRNA are in the human liver and kidney, with detectable expression in lung and heart. Therefore, this human 5-HT2B receptor could account for functions attributed to the peripheral 5-HT1D/5-HT2-like receptors, especially in the cardiovascular system. Thus, its detailed original pharmacology is of prime importance for therapeutic drug development (Choi, 1994b).

The recently characterized 5-HT2B subfamily of serotonin receptors has now been reported from three different species: human, rat and mouse. Their genomic structures include 2 introns present at identical positions. Despite this similarity, their respective protein sequences show some diversities. In addition, the pharmacology of these receptors is distantly related, and their sites of expression vary among species. Thus, it appears difficult at present to unambiguously classify these receptors into the same subfamily, raising the possibility of the existence of other 5-HT2B-like receptors, yet to be discovered (Choi, 1996b).

The 5-HT2B receptor is particularly interesting since it may be involved in diseases such as migraine. The isolation of a human 5-HT2B receptor clone from a cDNA library derived from SH-SY5Y cells is described. Although the receptor sequence was only 80% homologous to the rat sequence, the exon-intron distribution is conserved between the two species. In the human body, the receptor mRNA was detected in most peripheral organs. Only low expression levels are found in the brain. After expression in HEK 293 cells, activation of the receptor stimulates the production of phosphatidylinositol. The pharmacology of this functional response correlates well with that of the rodent receptor (Schmuck, 1994).

5-HT2B receptors, in addition to phospholipase C stimulation, are able to trigger activation of the proto-oncogene product p21ras. During mouse embryogenesis, a peak of 5-HT2B receptor expression is detected at the neurulation stage; 5-HT2B expression in neural crest cells, heart myocardium, and somites. The requirement for functional 5-HT2B receptors shortly after gastrulation, is supported by the culture of embryos exposed to 5-HT2B-high affinity antagonist such as ritanserin, which induces morphological defects in the cephalic region, heart and neural tube. Functional 5-HT2B receptors are also expressed during the serotonergic differentiation of the mouse F9 teratocarcinoma-derived clonal cell line 1C11. Upon 2 days of induction by cAMP, 5-HT2B receptors become functional, and on day 4, the appearance of 5-HT2A receptors coincides with the onset of active serotonin transporter by these cells. Active serotonin uptake is modulated by serotonin, suggesting autoreceptor functions for 5-HT2B receptors (Choi, 1998).

5-HT receptors: Protein interactions

Productive interaction between receptors and G proteins involves multiple intracellular receptor domains, but the role of individual receptor amino acids in directing the selection of specific signaling pathways has not yet been identified. Sequence alignment of several G protein-coupled receptors has identified a highly conserved threonine residue in the i2 loop of the 5-hydroxytryptamine 1A (5-HT1A) receptor that is a putative protein kinase C phosphorylation consensus site and is located in a predicted amphipathic alpha-helical domain. To examine the role of this conserved threonine residue in 5-HT1A receptor coupling to Gi/Go proteins, this residue was mutated to alanine (T149A mutant). Wild-type and mutant 5-HT1A receptors were stably transfected into both Ltk- and GH4C1 cells to investigate receptor coupling to multiple signaling pathways. In both cell lines, the T149A mutant displays similar agonist affinities as the wild-type receptor. In Ltk- cells, the T149A 5-HT1A receptor inhibits cAMP accumulation by 30%, when compared with wild-type (83%). A 2.6-fold increase in intracellular calcium (due to phospholipase C-mediated calcium mobilization) is observed for the wild-type receptor upon the addition of 100 nM 5-HT; whereas the T149A 5-HT1A receptor fails to mediate a calcium mobilization response at equivalent receptor levels to wild-type. When transfected in GH4C1 cells, the T149A receptor mutant fully inhibits basal cAMP and partially inhibits Gs-stimulated cAMP accumulation, as compared with wild-type receptor (57 +/- 14% versus 86 +/- 2%). In contrast, the T149A 5-HT1A receptor mutant fails to block the influx of calcium induced by calcium channel agonist (+/-)-Bay K8644, whereas the wild-type 5-HT1A receptor inhibits the calcium influx by 40%. Thus, the Thr149 residue is directly involved in G protein coupling to calcium mobilization (mediated by betagamma subunits of Gi2) and to inhibition of calcium channel activation (mediated by betagamma subunits of Go) but plays a minor role in coupling to alpha1-mediated inhibition of cAMP accumulation. The conserved i2 loop threonine may serve as a G protein contact site to direct the signaling specificity of multiple receptors (Lembo, 1997).

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) elicits a wide array of physiological effects by binding to several receptor subtypes. The 5-HT2 family of receptors belongs to a large group of seven-transmembrane-spanning G-protein-coupled receptors and includes three receptor subtypes [5-HT2A, 5-HT(2B) and 5-HT(2C)] that are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol. Transcripts encoding the 2C subtype of serotonin receptor [5-HT(2C)R] undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminase(s). Sequence analysis of complementary DNA isolates from dissected brain regions have indicated the tissue-specific expression of seven major 5-HT(2C) receptor isoforms encoded by eleven distinct RNA species. Editing of 5-HT(2C)R messenger RNAs alters the amino-acid coding potential of the predicted second intracellular loop of the receptor and can lead to a 10-15-fold reduction in the efficacy of the interaction between receptors and their G proteins. These observations indicate that RNA editing is a new mechanism for regulating serotonergic signal transduction and suggest that this post-transcriptional modification may be critical for modulating the different cellular functions that are mediated by other members of the G-protein-coupled receptor superfamily (Burns, 1997).

Effects of dopamine receptor antagonists

Serotonin-2 receptor antagonists, like ritanserin, greatly enhance deep slow wave sleep (SWS-2) and low-frequency EEG power in humans and rodents. 5-HT2A and 5-HT2C receptors may be involved in these effects, but the role of the 5-HT2B receptor is still unclear. To investigate the role of the 5-HT2B receptor in regulation of the sleep-wake cycle, the subtype-selective antagonist SB-215505 was administered to Sprague-Dawley rats at light onset (beginning of passive phase). EEG, EMG and motor activity were recorded during the subsequent 8 h. SB-215505 dose-dependently increased wakefulness (W) at the expense of the intermediate stage of sleep, paradoxical sleep (PS) and SWS-2 in the first hour. Parallel to increased W, significantly increased motor activity was found. Spectral analysis of the EEG in W showed a dose-dependent decrease in power density in the 3-8 Hz frequency range (maximum effect at 6 Hz). In light slow wave sleep and SWS-2, the drug reduced low-frequency (<8 Hz) EEG power, suggesting decreased sleep intensity after SB-215505 treatment. In PS, the drug dose-dependently decreased EEG power solely in the theta (6-9 Hz) band, primarily affecting the peak power value (7 Hz). The well-known SWS-2 enhancing effect of 5-HT2 receptor antagonists is mediated by 5-HT2A and/or 5-HT2C receptors. In contrast, blockade of 5-HT2B receptors increases motor activity and W along with decreased theta activity during W and PS. Activation of 5-HT2B receptors may contribute to initiation of sleep and to theta generation during W and PS under physiological conditions (Kantor, 2004).

Mutation of Serotonin receptors

Serotonin plays key roles in sleep-wakefulness regulation. Evidence indicates that 5-HT2 receptors are involved mainly in non-rapid eye movement sleep (NREMS) regulation and respiratory control. This study investigated the relative contribution of 5-HT2A, 5-HT2B, and 5-HT2C receptor subtypes to NREMS and breathing during sleep, using 5-HT2 subtype-selective ligands in wild-type (5-HT2A+/+) and knock-out (5-HT2A-/-) mice that do not express 5-HT2A receptors. Acute blockade of 5-HT2A receptors induced an increase in NREMS in 5-HT2A+/+ mice, but not 5-HT2A-/- mutants, which spontaneously expressed less NREMS than wild-type animals. In 5-HT2A+/+ mice, 5-HT2B receptor blockade produced a reduction of NREMS, whereas receptor activation induced an increase in this sleep stage. These effects were less pronounced in 5-HT2A-/- mice, indicating a lower sensitivity of 5-HT2B receptors in mutants, with no change in 5-HT2B mRNA. Blockade of 5-HT2C receptors had no effect on NREMS in both strains. In addition, an increase in EEG power density after sleep deprivation was observed in 5-HT2A+/+ mice but not in 5-HT2A-/- mice. Whole-body plethysmographic recordings indicated that 5-HT2A receptor blockade in 5-HT2A+/+ mice reduced NREMS apneas and bradypneas that occurred after sighs. In contrast, in 5-HT2A-/- mutants, NREMS apneas were not modified, and bradypnea after sighs were more pronounced. These results demonstrate that 5-HT exerts a 5-HT2B-mediated facilitation of NREMS, and an influence respectively inhibitory on NREMS and facilitatory on sleep apnea generation, via 5-HT2A receptors. Moreover, 5-HT2A gene knock-out leads to functional compensations yielding adaptive changes opposite to those caused by pharmacological blockade of 5-HT2A receptors in 5-HT2A+/+ mice (Popa, 2005).

Activation, internalization, and recycling of the serotonin 2A receptor by dopamine

Serotonergic and dopaminergic systems, and their functional interactions, have been implicated in the pathophysiology of various CNS disorders. This study used recombinant serotonin (5-HT) 2A (5-HT2A) receptors to further investigate direct interactions between dopamine and 5-HT receptors. Previous studies in Xenopus oocytes showed that dopamine, although not the cognate ligand for the 5-HT2A receptor, acts as a partial-efficacy agonist. At micromolar concentrations, dopamine also acts as a partial-efficacy agonist on 5-HT2A receptors in HEK293 cells. Like 5-HT, dopamine also induces receptor-internalization in these cells, although at significantly higher concentrations than 5-HT. Interestingly, if the receptors are first sensitized or 'primed' by subthreshold concentrations of 5-HT, then dopamine-induced internalization occurs at concentrations approximately 10-fold lower than when dopamine is used alone. Furthermore, unlike 5-HT-mediated internalization, dopamine-mediated receptor internalization, alone, or after sensitization by 5-HT, does not depend on PKC. Dopamine-internalized receptors recycle to the surface at rates similar to those of 5-HT-internalized receptors. These results suggest a previously uncharacterized role for dopamine in the direct activation and internalization of 5-HT2A receptors that may have clinical relevance to the function of serotonergic systems in anxiety, depression, and schizophrenia and also to the treatment of these disorders (Bhattacharyya, 2006)

5-HT1 and 5-HT2 receptors stimulate the Ras pathway

The family of serotonin 5-HT2 receptors stimulates the phospholipase C second messenger pathway via the alpha subunit of the Gq GTP-binding protein. Agonist stimulation of the 5-HT2B receptor subtype stably expressed in the mouse fibroblast LMTK- cell line causes a rapid and transient activation of the proto-oncogene product p21ras, as measured by an increase in GTP-bound Ras in response to serotonin. Furthermore, 5-HT2B receptor stimulation activates p42mapk/p44mapk (ERK2/ERK1) mitogen-activated protein kinases, as assayed by phosphorylation of myelin basic protein. Antibodies against p21ras, Galphaq, -beta, or -gamma2 subunits of the GTP-binding protein inhibit MAP kinase-dependent phosphorylation. The MAP kinase activation is correlated with a stimulation of cell division by serotonin. In addition to this mitogenic action, transforming activity of serotonin is mediated by the 5-HT2B receptor since its expression in LMTK- cells is absolutely required for foci formation and for these foci to form tumors in nude mice. Expression of the 5-HT2B receptor was detected in spontaneous human and Mastomys natalensis carcinoid tumors and, similar to the 5-HT2B receptor transfected cells, the Mastomys tumor cells are also responsive to serotonin with similar coupling to p21ras activation (Launay, 1996).

Serotonin (5-HT) is a potent mitogen in many cells types, an action that is frequently mediated through pertussis toxin-sensitive G proteins. Pharmacological inhibitors and dominant negative signaling constructs have been used to delineate elements that participate in the activation of MAPK, a growth-associated mitogen-activated protein kinase, by human G protein-coupled 5-HT1A receptor transfected into CHO-K1 cells in a stable manner. The activation pathway does not directly involve phorbol ester-sensitive protein kinase C types, but does require (1) pertussis toxin-sensitive G protein beta gamma-subunits; (2) a staurosporine- and genistein-sensitive protein kinase; (3) phosphoinositide-3'-kinase activity; (5) activation of Sos in a multimolecular complex that contains p46Shc, and p52Shc, and Grb2; (5) the GTPase p21Ras, and (6) the protein kinase p74Raf-1. These data demonstrate that the 5-HT1A receptor mediates MAPK activity by convergence upon a common activation pathway that is shared with receptor tyrosine kinases (Garnovskaya, 1996).

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin [5-hydroxytryptamine (5-HT)] 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation is attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation. This is because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src, but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase have no effect on 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis is required for 5-HT1A receptor-mediated Erk1/2 activation, a role for CAM in receptor endocytosis is postulated. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuates 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevents agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. It is concluded that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects CAM's role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2 (Della Rocca, 1999).

5-HT receptors and lipid signaling

Among immortalized teratocarcinoma-derived cells, the clone 1C11 is a committed precursor of the neuronal lineage. On day 2 of its serotoninergic differentiation, this clone expresses only one subtype of serotonin [5-hydroxytryptamine (5-HT)] receptor, which is functionally coupled to phosphatidylinositol hydrolysis. The identity of these receptors was established by comparing their properties with those of 5-HT2B receptors expressed by LMTK- fibroblasts stably transfected with the recently cloned murine cDNA NP75 (LM5 cells). In both cell types, the analysis agonist (125I-DOI) binding reveals the presence of a single class of sites; the affinity of these sites for agonist is one order of magnitude lower than that reported for 5-HT2A receptors. In 1C11 cells differentiated for 2 days, as well as in LM5 cells, DOI binding is decreased by nonhydrolyzable analogs of GTP, indicating that the 5-HT2B receptor is functionally coupled to a G protein. The DOI-induced increase of phosphoinositide hydrolysis, which is correlated with both GTPase activity and binding data, is mediated by a Gq protein. This work demonstrates that the 5-HT2B receptor is functionally expressed before complete serotoninergic differentiation of 1C11 cells. The inducible 1C11 clone thus provides an in vitro model to investigate the possible role of the 5-HT2B receptor in the expression of the serotoninergic phenotype (Loric, 1995).

The study of signaling cascades and of functional interactions between 5-hydroxytryptamine (5-HT) receptor pathways with heterogenous brain cell populations remains an arduous task. A serotonergic cell line was used to elucidate cross-talk between 5-HT receptors and to demonstrate the involvement of two 5-HT2 receptor subtypes in the regulation of 5-HT1B/1D function. The inducible 1C11 cell line has the unique property of acquiring within 4 days a complete serotonergic phenotype (1C11* cells), including three 5-HT receptors. 5-HT1B/1D and 5-HT2B receptors are expressed from day 2 of the serotonergic differentiation, while 5-HT2A receptors are induced at day 4. 5-HT2B receptors are coupled with the phospholipase A2 (PLA2)-mediated release of arachidonic acid (AA) and the activation of 5-HT2B receptors in 1C11*d2 cells inhibits the 5-HT1B/1D receptor function via a cyclooxygenase-dependent AA metabolite. At day 4, this 5-HT2B-mediated inhibition of the 5-HT1B/1D function can be blocked upon concomitant 5-HT2A activation, although a 5-HT2A/PLA2 positive coupling is evident. This suggests the existence in 1C11*d4 cells of pathway(s) for 5-HT2A receptors, distinct from PLC and PLA2. Finally, this study reveals the antagonistic roles of 5-HT2A and 5-HT2B receptors in regulating the function of 5-HT1B/1D, a receptor involved in neuropsychiatric disorders and migraine pathogenesis (Tournois, 1998).

The 5-Hydroxytryptamine (5-HT)2C receptor (originally known as the 5-HT1C receptor) is a member of the 5-HT2 subfamily of G protein coupled receptors, which is known to couple to phospholipase C. Within the 5-HT2 subfamily, only the 5-HT2C receptor also couples to inhibition of forskolin-stimulated cAMP production when expressed at high density in stably transformed AV12 cells. The 5-HT2C receptor couples with high efficacy to both phospholipase C as measured by IP3 (inositol 1,4,5-trisphosphate) production and to inhibition of forskolin-stimulated cAMP production. The 5-HT2A and 5-HT2B receptors, while coupling to phospholipase C with high affinity (EC50s of 19.24 nM +/- 6.44 and 1.24 nM +/- 0.136 respectively), do not decrease adenylyl cyclase activity. The 5-HT2C receptor actions in both systems show the expected pharmacology for the 5-HT2C receptor, e.g., mesulergine antagonizes the effects of 5-HT and spiperone does not. Preincubation of cells with PTX shows that the G protein coupling of the 5-HT2C receptor to phospholipase C is PTX insensitive, while the G protein coupling to inhibition of adenylyl cyclase is PTX sensitive, even with concentrations as low as 20 ng/ml of PTX. PTX pretreatment of the 5-HT2C bearing cells also unmasks a small stimulatory effect on adenylyl cyclase. When expressed at low density the 5-HT2C receptor potentiates forskolin-stimulated cAMP production by 2 fold while still maintaining its ability to enhance PI hydrolysis. A more modest potentiation of cAMP production is noted with low density expression of the 5-HT2B receptor. Thus the ability of the 5-HT2C receptor to interact with several effectors through at least two different G proteins is, in part, receptor subtype specific but also influenced by receptor density (Lucaites, 1996).

A variety of receptors coupled to GTP-binding regulatory proteins (G proteins) initiate signals that culminate in activation of the mitogen-activated protein kinases ERK1 and ERK2. Similarly, the human 5-HT1A receptor expressed in Chinese hamster ovary cells promotes activation of ERK1 and ERK2, but the pathway used does not conform entirely to those proposed previously for G protein-coupled receptors. Activation of ERK2 by the 5-HT1A receptor-selective agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin hydrobromide (8-OH-DPAT) is inhibited completely by pertussis toxin and substantially by prolonged treatment of cells with phorbol 12-myristate 13-acetate. However, the implied requirement for protein kinase C is negated in studies with bisindolylmaleimide and Ro-31-8220, which, although completely inhibiting activation of ERK2 by phorbol ester, has no impact on activation by 8-OH-DPAT. Moreover, the anticipated inhibition by the tyrosine kinase inhibitors genistein and herbimycin A is marginal at best. As expected for a Gi-coupled receptor, the inhibitors of phosphatidylinositol 3-kinase wortmannin and LY294002 inhibit activation of ERK2, albeit only partly (70%). Of significance, an inhibitor of a phosphatidylcholine-specific phospholipase C, tricyclodecan-9-yl-xanthogenate (D609), causes a similar degree of inhibition. When the two types of inhibitors are combined, an almost complete inhibition is achieved. These data suggest that phosphatidylinositol 3-kinase and phosphatidylcholine-specific phospholipase C represent components of different, but partly overlapping pathways that can account almost entirely for the activation of ERK2 by the 5-HT1A receptor (Cowen, 1996).

Chinese hamster ovary cells (CHO-K1) express an endogenous 5-hydroxytryptamine (5-HT)1B-like receptor that is negatively coupled to adenylyl cyclase through a pertussis toxin (PTX)-sensitive mechanism. Furthermore, the human adenosine A1 receptor, when expressed in CHO-K1 cells (CHO-A1), has been shown to mobilize intracellular Ca2+ through a PTX-sensitive mechanism. Therefore the aim of this investigation was to determine whether the endogenous 5-HT1B-like receptor is able to stimulate increases in intracellular free [Ca2+] ([Ca2+]i) in CHO-A1 cells. In agreement with previous studies using CHO cells, 5-hydroxytryptamine (5-HT) elicited a concentration-dependent inhibition of forskolin-stimulated [3H]-cyclic AMP production in CHO-A1 cells. 5-HT inhibits about half of the [3H]-cyclic AMP accumulation induced by 3 microM forskolin. Forskolin stimulated [3H]-cyclic AMP accumulation is also inhibited by 5-HT1 receptor agonists. 5-HT elicits a concentration-dependent increase in [Ca2+]i in CHO-A1 cells. In the presence of 2 mM extracellular Ca2+, 5-HT more than doubles [Ca2+]i. These data demonstrate that in CHO-K1 cells the endogenously expressed 5-HT1B-like receptor couples to the phospholipase C/Ca2+ signaling pathway through a PTX-sensitive pathway, suggesting the involvement of Gi/Go protein(s) (Dickenson, 1996).

Alzheimer's disease amyloid consists of amyloid beta-peptides (Abeta) derived from the larger precursor amyloid precursor protein (APP). Non-amyloidogenic APP processing involves regulated cleavage within the Abeta domain followed by secretion of the ectodomain (APPs). APPs secretion can be stimulated by muscarinic acetylcholine receptors coupled to phospholipases and kinases. To determine whether other receptor classes can regulate APP processing, the relation between serotonin receptors and APPs secretion was examined. Serotonin increases APPs release 3-4-fold in 3T3 cells that stably overexpress either 5-HT2aR or 5-HT2cR. The increase is dose-dependent and is blocked by serotoninergic antagonists. Phorbol esters also increase APPs secretion, but neither kinase inhibitors nor down-regulation of PKC block the serotonin-induced increase in APPs secretion. Thus PKC is not necessary to stimulate APPs secretion. Phospholipase A2 (PLA2) inhibitors block the 5-HT2aR-mediated increase in APPs secretion, suggesting a role for PLA2 in coupling 5-HT2aR to APP processing. In contrast, coupling of 5-HT2cR to APPs secretion involves both PKC and PLA2. Serotonin also stimulates the release of the APLP2 ectodomain, suggesting that additional members of the APP multigene family are processed via similar regulated pathways. Inasmuch as generation of APPs precludes the formation of amyloidogenic derivatives, serotonin receptors provide a novel pharmacological target to reduce these derivatives in Alzheimer's disease (Nitsch, 1996).

Serotonin (5-hydroxytryptamine; 5-HT) 5-HT2A and 5-HT2C receptors belong to the class of phosphoinositide-specific phospholipase C (PLC)-linked receptors. Conditions were established for measuring 5-HT2A-linked and 5-HT2C-linked PLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca2+ (300 nM) and GTPgammaS (1 microM), 5-HT increases PLC activity in caudate membranes. Pharmacological analysis using the selective 5-HT2A antagonist (spiperone), and the nonselective 5-HT(2A/2C) antagonist (mianserin), demonstrates that over half of the 5-HT-stimulated PLC activity is due to stimulation of 5-HT2C receptors as opposed to 5-HT2A receptors. Radioligand binding assays with [3H]RP 62203 and [3H]mesulergine were used to quantify 5-HT2A and 5-HT2C sites, respectively, in caudate. In contrast to activity in caudate, PLC activity in frontal cortex is stimulated by 5-HT in a manner that is inhibited by the 5-HT2A-selective antagonists: spiperone and ketanserin. Taken together, the results indicate that 5-HT2A- and 5-HT2C-linked PLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5-HT2C-mediated phosphoinositide hydrolysis is observed in caudate, despite the relatively low density of 5-HT2C sites. The significance of these observations with respect to the physiological function of 5-HT2C receptors is discussed (Wolf, 1997).

To investigate the receptor-channel coupling pathway, the coding region of the 5-HT1a receptor was subcloned into two plasmid vectors pSP64(polyA+) and pSP64T. Compared to the original 5-HT1a receptor construct G-21, both new constructs greatly increase the expression of functional 5-HT1a receptors in Xenopus oocytes, which develop large inward current responses to 5-HT. These responses are dose-dependent (EC50 approximately 150 nM), and can be elicited also by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The 5-HT1a receptor mediated current has an oscillatory time course, and a reversal potential close to the equilibrium potential for Cl- (ca. -25 mV). Moreover, during and for some minutes following the application of 5-HT, these oocytes acquire the property of generating a transient inward current when their membrane is hyperpolarized. These features are characteristic of responses mediated by other receptors (e.g. muscarinic, angiotensin, serum receptors, etc.) that are known to couple to the endogenous PLC/PI second messenger pathway in Xenopus oocytes. In particular, the 5-HT1a receptor mediated current is very similar to the current induced by 5-HT-stimulation of heterogenic 5-HT2c receptors. These results show further that the 5-HT1a receptor couples to the endogenous PLC/PI pathway much less efficiently than the 5-HT2c receptor. These results demonstrate clearly that the human 5-HT1a receptor can couple efficiently to the Xenopus oocyte endogenous PLC/PI pathway, and provide additional evidence for cell-specific signal transduction (Ni, 1997).

There are many examples of a single receptor coupling directly to more than one cellular signal transduction pathway. Although traditional receptor theory allows for activation of multiple cellular effectors by agonists, it predicts that the relative degree of activation of each effector pathway by an agonist (relative efficacy) must be the same. Agonists at the human serotonin2A (5-HT2A) and 5-HT2C receptors activate differentially two signal transduction pathways independently coupled to the receptors [phospholipase C (PLC)-mediated inositol phosphate (IP) accumulation and phospholipase A2 (PLA2)-mediated arachidonic acid (AA) release]. The relative efficacies of agonists differ depending on which signal transduction pathway is measured. Moreover, relative to 5-HT, some 5-HT2C agonists (e.g., 3-trifluoromethylphenyl-piperazine) preferentially activate the PLC-IP pathway, whereas others (e.g., lysergic acid diethylamide) favor the PLA2-AA pathway. In contrast, when two dependent responses are measured (IP accumulation and calcium mobilization), agonist relative efficacies are not different. These data strongly support the hypothesis termed 'agonist-directed trafficking of receptor stimulus'. Concentration-response curves to 5-HT2C agonists were fit well by a three-state model of receptor activation, suggesting that two active receptor states may be sufficient to explain pathway-dependent agonist efficacy. Rational drug design that optimizes preferential effector activity within a group of receptor-selective drugs holds the promise of increased selectivity in clinically useful agents (Berg, 1998a).

There is now considerable evidence that a single receptor subtype can couple to multiple effector pathways within a cell. The concept termed 'agonist-directed trafficking of receptor stimulus' suggests that agonists may be able to selectively activate a subset of multiple signaling pathways coupled to a single receptor subtype. 5-HT2A and 5-HT2C receptors couple to phospholipase C-(PLC) mediated inositol phosphate (IP) accumulation and PLA2-mediated arachidonic acid (AA) release. Relative efficacies of agonists (referenced to 5-HT) differ depending on whether IP accumulation or AA release is measured. For the 5-HT2C receptor system, some agonists (e.g. TFMPP) preferentially activate the PLC-IP pathway, whereas others (e.g. LSD) favored PLA2-AA. As expected, the EC50s of agonists do not differ between pathways. For the 5-HT2A receptor system, all agonists tested had greater relative efficacy for PLA2-AA than for PLC-IP. In contrast, relative efficacies were not different for 5-HT2A agonists when sequential effects in a pathway were measured (IP accumulation vs. calcium mobilization). These data strongly support the agonist-directed trafficking hypothesis (Berg, 1998b).

Mechanisms underlying the 5-HT2A receptor induction of intracellular Ca2+ mobilization and Ca2+ influx in type I astroglial cells in primary culture from newborn rat cerebral cortex were evaluated. The 5-HT-evoked Ca(2+)-transients, inhibited by the 5-HT2A antagonists ketanserin or 4-(4-fluorobenzoyl)-1-(4-phenylbutyl) piperidine oxalate, consist of an initial peak caused by inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from internal stores, and a second sustained part which was due to Ca2+ transport over the plasma membrane. The responses are pertussis toxin-insensitive, suppressed by the phospholipase C inhibitor neomycin and were inhibited by the Ca(2+)-ATPase inhibitor thapsigargin. Furthermore, the responses are inhibited by the IP3 receptor antagonist heparin. When the second sustained part of the 5-HT-evoked response was studied, it was concluded that Ca2+ influx is not a result of opening of voltage operated calcium channels of either L, N or T-type. Instead it appeared that Ca2+ enters the cells through specialized voltage independent Ca2+ channels, which are dependent on IP3 production and subsequent Ca2+ release from internal stores. From this, it is concluded that 5-HT opens Ca2+ channels in astrocytes that closely resemble depletion-operated Ca2+ channels (DOCCs) (Hagberg, 1998).

Signaling pathways responsible for serotonin (5-HT)-mediated induction of early response genes prostaglandin G/H synthase-2 (PGHS-2, cyclooxygenase-2) and egr-1 were investigated in rat mesangial cells. Gene induction by 5-HT is dependent on 5-HT2A receptors that are pertussis toxin insensitive, which indicates coupling to a G-protein of the Gq family. Binding of 5-HT to this receptor activates phosphatidylinositol-specific phospholipase C (PLC) and release of Ca2+ from internal stores, but this activation is not related to PGHS-2 mRNA expression. Similarly, PI-3 kinase is not involved in 5-HT signaling. Instead, inhibition of phosphatidylcholine-specific PLC interfers with PGHS-2 and egr-1 mRNA induction, suggesting this enzyme is a link between 5-HT2A receptors and protein kinase C, an essential part of 5-HT-mediated signaling. The MAP kinase pathway has been identified as common signaling pathway of 5-HT or phorbol ester-induced gene expression. Increase of intracellular cAMP by forskolin or dibutyryl cAMP does not induce PGHS-2 or egr-1 mRNA expression by itself, but strongly inhibits 5-HT-mediated mRNA induction. PGHS-2 mRNA and protein induction by 5-HT is also abolished by chelation of Ca2+ ions by EGTA, suggesting involvement of Ca2+-dependent enzymes. In contrast, egr-1 mRNA expression is superinduced in the presence of EGTA. Induction of Egr-1 protein is not changed by EGTA; this hints at possible Ca2+ sensitive posttranscriptional steps. Activation of the Gq-coupled 5-HT2A receptor thus leads to the expression of the early response genes PGHS-2 and egr-1, using common as well as differing signaling elements that allow differential regulation of the expression of these genes that are functionally related to renal hemodynamics and proliferation of mesangial cells, respectively (Goppelt-Struebe, 1998).

Miscellaneous 5-HT receptor functions that do not involve lipid signaling

The action of 5-hydroxytryptamine (5-HT) via the 5-HT1A receptor on dissociated rat dorsal raphe neurons was characterized under the whole-cell mode by using the nystatin-perforated patch-clamp technique. Under voltage-clamp conditions, 5-HT induces an inwardly rectifying K+ current (I5-HT) in a concentration-dependent manner. I5-HT is mimicked by 5-HT1A receptor agonists. I5-HT is reversibly blocked by such 5-HT1A receptor antagonists. I5-HT is antagonized in a concentration-dependent manner by such K+ channel blockers as quinine, Ba2+ and 4-aminopyridine but is relatively insensitive to both CS+ and tetraethylammonium. When the neurons are loaded with guanosine 5'-O-3-thiotriphosphate through a patch pipette, the K+ current induced by 5-HT becomes irreversible. N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, irreversibly blocks I5-HT. The intracellular perfusion with a Ca2+ chelator, or neomycine (a phospholipase C inhibitor), does not significantly affect the 5-HT-induced response. A protein kinase C (PKC) activator has only a weak inhibitory effect on I5-HT, and a PKC inhibitor fails to significantly occlude I5-HT. Therefore, the K+ conductance activated via the 5-HT1a receptor of dorsal raphe neurons is thus characterized by the sensitivity to such K+ channel blockers as quinine, Ba2+ and 4-aminopyridine. Moreover, a G protein that is NEM-sensitive and can couple to the 5-HT1A receptor, is thus considered to activate the inwardly rectifying K+ conductance without being mediated by such second messengers as Ca2+ and PKC (Katayama, 1997).

The neurotransmitter serotonin mediates a wide variety of peripheral and central physiological effects through the binding to multiple receptor subtypes. Among them, serotonin 5-HT2A receptors are known to activate the phospholipase C-beta second messenger pathway. In rat skeletal muscle myoblasts a functional serotonin 5-HT2A receptor has been identified and localized. This receptor is detected on the plasma membrane, in myoblasts, and at the level of T-tubules in contracting myotubes. Binding of serotonin to its receptor increases the expression of genes involved in myogenic differentiation. Unexpectedly, the 5-HT2A receptor is able to activate another signaling pathway; it triggers a rapid and transient tyrosine phosphorylation of Jak2 kinase in response to serotonin. Jak2 auto-phosphorylation is followed by the tyrosine phosphorylation of STAT3 (signal transducers and activators of transcription) and its translocation into the nucleus. The 5-HT2A receptor and STAT3 co-precipitate with Jak2, indicating that they are physically associated. It is concluded that the serotonin 5-HT2A receptor identified in skeletal muscle myoblasts is able to activate the intracellular phosphorylation pathway used by cytokines. The presence of serotonin receptors in T-tubules suggests a role for serotonin in excitation-contraction coupling and/or as an effectof skeletal muscle fiber repair (Guillet-Deniau, 1997).

p90 ribosomal S6 kinase 2 exerts a tonic brake on the 5-HT2A receptor

G protein-coupled receptors (GPCRs) are essential for normal central CNS function and represent the proximal site(s) of action for most neurotransmitters and many therapeutic drugs, including typical and atypical antipsychotic drugs. Similarly, protein kinases mediate many of the downstream actions for both ionotropic and metabotropic receptors. Genetic deletion of p90 ribosomal S6 kinase 2 (RSK2; see Drosophila RSK) potentiates GPCR signaling. Initial studies of 5-hydroxytryptamine (5-HT)2A receptor signaling in fibroblasts obtained from RSK2 wild-type (+/+) and knockout (-/-) mice showed that 5-HT2A receptor-mediated phosphoinositide hydrolysis and both basal and 5-HT-stimulated extracellular signal-regulated kinase 1/2 phosphorylation are augmented in RSK2 knockout fibroblasts. Endogenous signaling by other GPCRs, including P2Y-purinergic, PAR-1-thrombinergic, beta1-adrenergic, and bradykinin-B receptors, was also potentiated in RSK2-deficient fibroblasts. Importantly, reintroduction of RSK2 into RSK2-/- fibroblasts normalized signaling, thus demonstrating that RSK2 apparently modulates GPCR signaling by exerting a 'tonic brake' on GPCR signal transduction. These results imply the existence of a novel pathway regulating GPCR signaling, modulated by downstream members of the extracellular signal-related kinase/mitogen-activated protein kinase cascade. The loss of RSK2 activity in humans leads to Coffin-Lowry syndrome, which is manifested by mental retardation, growth deficits, skeletal deformations, and psychosis. Because RSK2-inactivating mutations in humans lead to Coffin-Lowry syndrome, these results imply that alterations in GPCR signaling may account for some of its clinical manifestations (Sheffler, 2006).

5-HT receptor functions and the circadian response

In mammals, the environmental light/dark cycle strongly synchronizes the circadian clock within the suprachiasmatic nuclei (SCN) to 24 hr. It is well known that not only photic but also nonphotic stimuli can entrain the SCN clock. Actually, many studies have shown that a daytime injection of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH DPAT), a serotonin 1A/7 receptor agonist, as a nonphotic stimulus induces phase advances in hamster behavioral circadian rhythms in vivo, as well as the neuron activity rhythm of the SCN in vitro. Recent reports suggest that mammalian homologs of the Drosophila clock protein Period are involved in photic entrainment. Therefore, an examination was made to determine whether phase advances elicited by 8-OH DPAT are associated with a change of Period mRNA levels in the SCN. In this experiment, partial cDNAs were cloned encoding hamster Per1, Per2, and Per3 and both circadian oscillation and the light responsiveness of Period were observed. The inhibitory effect of 8-OH DPAT on hamster Per1 and Per2 mRNA levels in the SCN occurs only during the hamster's mid-subjective day, but not during the early subjective day or subjective night. The present findings demonstrate that the acute and circadian time-dependent reduction of Per1 and/or Per2 mRNA in the hamster SCN by 8-OH DPAT is strongly correlated with the phase resetting in response to 8-OH DPAT (Horikawa, 2000).

Exposure to UV radiation induces skin cancer and suppresses the immune response. To induce immune suppression, the electromagnetic energy of UV radiation must be absorbed by an epidermal photoreceptor and converted into a biologically recognizable signal. Two photoreceptors have been recognized: DNA and trans-urocanic acid (UCA). Trans-UCA is normally found in the outermost layer of skin and isomerizes to the cis isomer upon exposure to UV radiation. Although UCA was identified as a UV photoreceptor years ago, and many have documented its ability to induce immune suppression, its exact mode of action remains elusive. Particularly vexing has been the identity of the molecular pathway by which cis-UCA mediates immune suppression. This study provides evidence that cis-UCA binds to the serotonin [5-hydroxytryptamine (5-HT)] receptor with relatively high affinity (Kd = 4.6 nM). Anti-cis-UCA antibody precipitates radiolabeled 5-HT, and the binding is inhibited by excess 5-HT and/or excess cis-UCA. Similarly, anti-5-HT antibody precipitates radiolabeled cis-UCA, and the binding is inhibited by excess 5-HT or excess cis-UCA. Calcium mobilization is activated when a mouse fibroblast line, stably transfected with the human 5-HT2A receptor, is treated with cis-UCA. Cis-UCA-induced calcium mobilization os blocked with a selective 5-HT2A receptor antagonist. UV- and cis-UCA-induced immune suppression is blocked by antiserotonin antibodies or by treating the mice with 5-HT2A receptor antagonists. These findings identify cis-UCA as a serotonin receptor ligand and indicate that the immunosuppressive effects of cis-UCA and UV radiation are mediated by activation of the 5-HT2A receptor (Walterscheid, 2006).

Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice

Serotonin neurotransmission in the central nervous system modulates depression and anxiety-related behaviors in humans and rodents, but the responsible downstream receptors remain poorly understood. Global disruption of 5-HT2A receptor (5HT2AR) signaling in mice reduces inhibition in conflict anxiety paradigms without affecting fear-conditioned and depression-related behaviors. Selective restoration of 5HT2AR signaling to the cortex normalizes conflict anxiety behaviors. These findings indicate a specific role for cortical 5HT2AR function in the modulation of conflict anxiety, consistent with models of cortical, 'top-down' influences on risk assessment (Weisstaub, 2006).

Endogenous 5-HT2B receptor activation regulates neonatal respiratory activity in vitro

An function for 5-HT2B receptors in central nervous system has not yet been clearly elucidated. The role of different 5-HT(2) receptor subtypes has been studied in the medullary breathing center, the pre-Botzinger complex, and on hypoglossal motoneurons in rhythmically active transversal slice preparations of neonatal rats and mice. Local microinjection of 5-HT(2) receptor agonists revealed tonic excitation of hypoglossal motoneurons. Excitatory effects of the 5-HT2B receptor agonist BW723C86 could be blocked by bath application of LY272015, a highly selective 5-HT2B receptor antagonist. Excitatory effects of the 5-HT(2A/B/C) receptor agonist alpha-methyl 5-HT could be blocked by the preferential 5-HT2A receptor antagonist ketanserin. Therefore, 5-HT-induced excitation of hypoglossal motoneurons is mediated by convergent activation of 5-HT2A and 5-HT2B receptors. Local microinjection of BW723C86 in the pre-Botzinger complex increased respiratory frequency. Bath application of LY272015 blocked respiratory activity, whereas ketanserin had no effect. Therefore, endogenous 5-HT appears to support tonic action on respiratory rhythm generation via 5-HT2B receptors. In preparations of 5-HT2B receptor-deficient mice, respiratory activity appeared unaltered. Whereas BW723C86 and LY272015 had no effects, bath application of ketanserin disturbed and blocked rhythmic activity. This demonstrates a stimulatory role of endogenous 5-HT2B receptor activation at the pre-Botzinger complex and hypoglossal motoneurons that can be taken up by 5-HT2A receptors in the absence of 5-HT2B receptors. The presence of functional 5-HT2B receptors in the neonatal medullary breathing center indicates a potential convergent regulatory role of 5-HT2B and -2A receptors on the central respiratory network (Gunther, 2006).

Developmental role of Serotonin and 5-HT2B receptors

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).

Modulation of dopamine release by striatal 5-HT2C receptors

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).

Ecstasy-induced cell death in cortical neuronal cultures is serotonin 2A-receptor-dependent and potentiated under hyperthermia

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).

Seratonin and liver regeneration

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)

Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus

Serotonin (5-HT) is a neuromodulator that plays many different roles in adult and embryonic life. Among the 5-HT receptors, 5-HT2B is one of the key mediators of 5-HT functions during development. Xenopus laevis was used as a model system to further investigate the role of 5-HT2B in embryogenesis, focusing on craniofacial development. By means of gene gain- and loss-of-function approaches and tissue transplantation assays, it was demonstrated that 5-HT2B modulates, in a cell-autonomous manner, postmigratory skeletogenic cranial neural crest cell (NCC) behavior without altering early steps of cranial NCC development and migration. 5-HT2B overexpression induced the formation of an ectopic visceral skeletal element and altered the dorsoventral patterning of the branchial arches. Loss-of-function experiments revealed that 5-HT2B signaling is necessary for jaw joint formation and for shaping the mandibular arch skeletal elements. In particular, 5-HT2B signaling is required to define and sustain the Xbap expression necessary for jaw joint formation. To shed light on the molecular identity of the transduction pathway acting downstream of 5-HT2B, the function was analyzed of phospholipase C beta 3 (PLC) in Xenopus development, and it was shown that PLC is the effector of 5-HT2B during craniofacial development. These results unveiled an unsuspected role of 5-HT2B in craniofacial development and contribute to understanding of the interactive network of patterning signals that is involved in the development and evolution of the vertebrate mandibular arch (Reisoli, 2010).

It is tempting to speculate that endothelin receptor and serotonergic receptor 2B signaling cooperate in reinforcing the PLC pathway and in modulating Xbap expression during first arch NCC patterning. However, although endothelin signaling is able to modulate both Xbap and XHand2 gene expression, 5-HT2B signaling seems to be required to maintain Xbap expression in the domain of the mandibular arch that gives rise to the jaw joint. In 5-HT2B morphants, in fact, XHand2 mRNA expression is unaltered and, consequently, the distal part of Meckel's cartilage develops normally. As the regulatory network involved in the specific expression of Bapx1 in vertebrates is conserved, perhaps 5-HT2B signaling controls Bapx1 expression in mammalian embryos as well. This aspect, to be verified and further explored, could be of interest particularly in the light of the possible involvement of BAPX1 in human birth defects, such as those of the oculo-articular-vertebral spectrum, which involve alterations in the first and second branchial arch derivatives, and considering the wide spectrum of serotonergic drugs available and commonly used in therapy including during pregnancy (Reisoli, 2010).


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date revised: 22 January 2017

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