InteractiveFly: GeneBrief

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

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-HT_1A-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-HT_1A-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-HT_1BDro 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-HT_1A/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-HT_1A-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-HT_1A/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-HT_1A/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-HT_1A-like receptors are also expressed presynaptically and are predicted to have autoreceptor function. The data indicate that administration of the 5-HT_1A receptor agonist U92016A in combination with the 5-HT_1A receptor antagonist WAY100635 work synergistically to attenuate STM function in the fly. Although these agents are both targeting 5-HT_1A 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-HT_1A-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-HT_1A antagonists in combination with 5-HT₂ antagonists are interfering, indicating a functional interaction between the two receptor circuitries. In mammals, 5-HT_1A 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-HT_1ADro 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-HT_1A 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-HT_1A-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).

CREBB repression of protein synthesis in mushroom body gates long-term memory formation in Drosophila

Learned experiences are not necessarily consolidated into long-term memory (LTM) unless they are periodic and meaningful. LTM depends on de novo protein synthesis mediated by cyclic AMP response element-binding protein (CREB) activity. In Drosophila, two creb genes (crebA, crebB) and multiple CREB isoforms have reported influences on aversive olfactory LTM in response to multiple cycles of spaced conditioning. How CREB isoforms regulate LTM effector genes in various neural elements of the memory circuit is unclear, especially in the mushroom body (MB), a prominent associative center in the fly brain that has been shown to participate in LTM formation. This study reports that 1) spaced training induces crebB expression in MB α-lobe neurons and 2) elevating specific CREBB isoform levels in the early α/β subpopulation of MB neurons enhances LTM formation. By contrast, learning from weak training 3) induces 5-HT1A serotonin receptor synthesis, 4) activates 5-HT1A in early α/β neurons, and 5) inhibits LTM formation. 6) LTM is enhanced when this inhibitory effect is relieved by down-regulating 5-HT1A or overexpressing CREBB. These findings show that spaced training-induced CREBB antagonizes learning-induced 5-HT1A in early α/β MB neurons to modulate LTM consolidation (Lin, 2022).

Recurrent spaced learning has been shown to relieve inhibition and gate LTM formation in animal models. However, gene regulatory mechanisms that act to filter relevant signals of repeated events and override inhibitory constraints in identified circuit elements remain unknown. The current data suggest that MB neurons in Drosophila provide a compelling cellular gating mechanism for LTM formation. Weak learning is sufficient to increase 5-HT1A synthesis in early α/β neurons, and these neurons produce a downstream inhibitory effect on LTM formation. After spaced training, CREBB expression represses further 5-HT1A synthesis, thereby relieving the inhibitory effect on LTM formation. These conclusions are supported by several lines of evidence: i) CREBB transcription increased after 5xS or 10xS but not after 1x (Fig. 1); and ii) RNAi-mediated knockdown of CREBB in α/β impaired LTM (Fig. 1), while overexpression of a crebB-a or crebB-c transgene enhanced LTM. iii) Conversely, RNAi-mediated knockdown of 5-HT1A in early α/β neurons enhanced LTM, while overexpression of a 5-HT1A transgene impaired LTM; and iv) 1x was sufficient to activate 5-HT1A, and this activation was inhibited by expression of CREBB proteins. v) Furthermore, overexpression of 5-HT1A-mediated LTM impairment was fully rescued by CREBB overexpression. Together, these findings suggest that synthesis of 5-HT1A and CREBB proteins in response to training operate like an opposing molecular switch to inhibit or disinhibit downstream LTM formation, respectively (Lin, 2022).

Previous reports suggested that expression of a chimeric CREBB-a transcriptional activator and a CREBB-b transcriptional repressor throughout whole fly enhanced and impaired LTM formation, respectively. Subsequently, CREBB-a-dependent enhancement of LTM was not observed using a hs-Gal4 driver that has low expression in MB. Chronic expression of a CREBB-b in all α/β neurons was shown to impair 1-d memory after spaced training. It has been documented, however, that these chronic disruptions of CREBB-b produced developmental abnormalities in MB structure. In contrast, acute induced expression of CREBB-b only in adult α/β neurons did not impair 1-d memory after spaced training (and did not produce structural defects). Using a different inducible system (MB247-Switch) to acutely expresses CREBB-b in γ and α/β neurons showed a mild impairment of 1-d memory after spaced training. More interestingly, various molecular genetic tools were used to show that interactions among CREBB, CREB-binding protein, and CREB-regulated transcription coactivator in MB were clearly involved in LTM formation or maintenance, respectively. Using the same inducible gene switch tool, a positive regulatory loop has been shown between Fos and CREBB in MB during LTM formation - but that study did not show behavioral data pertaining to manipulation of CREBB per se - nor did that study restrict experiments to early α/β neurons (Lin, 2022).

Zhang (2015) expressed a CRE-luciferase transgene in different subpopulations of MB neurons and then monitored luciferase activity in live flies at various times after spaced training. Immediately after spaced training, some patterns of luciferase expression decreased (OK107 expressing in all MB neurons; c739 expressing in all α/β neurons; 1471 expressing in γ neurons), or increased (c747 and c772 expressing variably in all MB neurons), or showed no detectable change (c320 expressing variably in γ, α'/β' and α/β subpopulation, 17d expressing primarily in late α/β and in early α/β neurons). Indeed, the Zhang paper pointed out that, because the CRE-reporter was expressed in more than one subpopulation of MB neurons, only net effects of CREB function could be quantified. Furthermore, this study did not elucidate which CREBB isoforms might increase or decrease after spaced training. Obviously, this information would be critical if different isoforms have opposing activator and repressor functions in specific MB neuron subpopulations. The current study provides a dramatic example of this point. By restricting manipulation to early α/β neurons in adult stage animals, this study showed that enhanced LTM formation after acute CREBB-c overexpression is comparable to the net effect of chimeric CREBB-a overexpression in whole flies, and that spaced training serves to increase the expression of CREBB in these early α/β neurons (Lin, 2022).

Yin (1995) reported that the CREBB-a isoform functions as a PKA-responsive transcriptional activator and the CREBB-b isoform functions as a repressor of CREBB-a-induced gene activation. Using new KAEDA synthesis as a reporter for temporal gene activation, it has been previously shown that CREBB-b in DAL neurons represses CREBA-mediated gene activation to inhibit LTM formation. In early α/β MB neurons, KAEDA experiments indicate that CREBB-a and CREBB-c, but not CREBB-b, both repress 5-HT1A-mediated inhibition to gate LTM formation. These findings demonstrate a neuron- and training-specific CREBA activation and CREBB repression of effecter genes involved in modulating LTM formation. Although crebB promoter-driven Gal4 expression, crebBRNAi downregulation, and cell-type specific transcriptomes show CREBB expression in early α/β neurons, it remains unclear whether specific naturally occurring CREBB isoforms in these neurons serve to modulate LTM formation (Lin, 2022).

How is the learning-induced LTM gating mechanism differentially regulated by different [1x, 10xM (ten massed cycles of training without rest intervals) or 10xS (spaced trials)] training protocols? Expression of both 5-HT1A and crebB in early α/β MB neurons was elevated 24 h after 10xS, whereas only 5-HT1A was induced after 1x, and neither gene was induced after 10xM. Why is elevated 5-HT1A seen after 10xS, when constitutive expression of CREBB proteins suppresses 5-HT1A expression? A possible explanation is that 5-HT1A may be normally activated as an early response to 1x, whereas crebB induction by 10xS is not evident for about 3 h. Gradual cessation of 5-HT1A transcription by the delayed 10xS-induced CREBB expression may account for lower KAEDE levels observed in one odor/shock pairing experiment. Interestingly, the data showed that even with elevated 5-HT1A, CREBB proteins can still enhance 1-d memory, suggesting that CREBB-mediated inhibition is rather complex (Lin, 2022).

Massed training appears not to activate or suppress learning-induced transcriptional activity in early α/β neurons, and 5-HT1A nor crebB is activated after 10xM. Nevertheless, massed training may antagonize LTM formation. For instance, in MB neurons, spaced training induces repetitive waves of Ras/mitogen-activated protein kinase (MAPK) activity, activates MAPK translocation to the nucleus mediated by importin-7 (29), increases CREBB expression and, in dorsal-anterior-lateral (DAL) neurons, training induces activity-dependent crebA, CamKII, and per gene expression - all of which are not activated after massed training. These notions above suggest that massed training produces a more upstream general suppression of these 1x- and 10xS-induced genes required for inhibitory/gating mechanisms allocated in MB and DAL neurons, respectively (Lin, 2022).

An LTM enhancing role associated with CREBB expression and protein synthesis inhibition is a novel aspect of this gating mechanism. A previous study showed that inhibition of protein synthesis in MB after strong spaced training did not reduce LTM. Since it would not be possible to detect enhanced performance in these experiments, the possibility cannot be excluded that this inhibition might eliminate downregulation of LTM effector genes, with a net effect of promoting the formation of LTM rather than impairing it. This study estalished that synthesis of new 5-HT1A proteins in early α/β neurons after weak learning provides negative regulation and produces a downstream inhibitory effect on LTM formation. Surprisingly, CREBB protein synthesis in early α/β neurons after strong spaced training provides positive regulation by antagonizing this negative effect of 5-HT1A on LTM . Thus, CREBB-mediated repression is equivalent to the net effect of blocking protein synthesis in MB. Both relieve downstream inhibition and enhance rather than impair LTM formation. It is proposed that CREBB-mediated inhibition operates both directly by repressing gene transcription and indirectly through activating their downstream translational suppression (Lin, 2022).

Together, these experiments uncover a biochemical LTM gating mechanism that requires delicate regulation of protein synthesis and repression after training within identified neurons. More broadly, these observations also highlight the need to confirm the regulatory functions of specific CREB isoforms in identified neuronal subtypes before making conclusions about their roles in LTM formation (Lin, 2022).

The discovery that molecules in early α/β neurons inhibit LTM formation is relevant to future studies. Another persistent anesthesia-resistant form of memory (ARM) is also mediated by α/β neurons and has been shown to inhibit LTM formation. 5-HT1A appears to be a key protein involved in both ARM and LTM. Furthermore, the interaction of serotonin released from dorsal paired medial neurons and 5-HT1A in α/β neurons is necessary for sleep. CREBB expression in MB is also under circadian regulation, which together suggests mechanistic links between ARM, LTM, sleep, and circadian timing in early α/β neurons (Lin, 2022).

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 [5HT_Dro7 and 5HT_Dro1A]) 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 5HT_7Dro receptor raises cAMP levels in cultured cells by activating adenylate cyclase. When 5HT_7Dro 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-5HT_7Dro and heat-shocked wild-type or parental lines) show no response to 5HT. Furthermore, there is no detectable impact on cGMP levels when 5HT_7Dro 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 c42-5HT_7Dro and c724-5HT_7Dro tubules appear to be additive: the sum of maximal rates is approximately equal to the maximal 5HT-induced rate observed in heat-shocked hs-5HT_7Dro 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 5HT_7Dro was investigated. In principal cells of 5HT_7Dro/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 5HT_7Dro/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-5HT_7Dro tubules (Kerr, 2004).

Another Drosophila 5HT receptor, 5HT_1ADro, is known to mobilize intracellular calcium. Expression of 5HT_1ADro 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 5HT_1ADro 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 K_i (18 μM) obtained for this receptor in cell lines (Kerr, 2004).

The 5HT_1ADro receptor has been previously shown to inhibit adenylate cyclase in vitro. Accordingly, cAMP and cGMP were measured in 5HT-stimulated c42-5HT_1ADro and c724-5HT_1ADro tubules. Activation of the 5HT_1ADro 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 5HT_Dro1A, expression has only been documented in some cells of the embryonic nervous system; 5HT_Dro7 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-HT^2A and 5-HT_2C receptors may be involved in these effects, but the role of the 5-HT_2B receptor is still unclear. To investigate the role of the 5-HT_2B 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-HT_2A and/or 5-HT_2C receptors. In contrast, blockade of 5-HT_2B receptors increases motor activity and W along with decreased theta activity during W and PS. Activation of 5-HT_2B 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-HT_2A, 5-HT_2B, and 5-HT_2C receptor subtypes to NREMS and breathing during sleep, using 5-HT2 subtype-selective ligands in wild-type (5-HT_2A+/+) and knock-out (5-HT_2A^-/-) mice that do not express 5-HT_2A receptors. Acute blockade of 5-HT_2A receptors induced an increase in NREMS in 5-HT_2A+/+ mice, but not 5-HT_2A^-/- mutants, which spontaneously expressed less NREMS than wild-type animals. In 5-HT_2A+/+ mice, 5-HT_2B receptor blockade produced a reduction of NREMS, whereas receptor activation induced an increase in this sleep stage. These effects were less pronounced in 5-HT_2A^-/- mice, indicating a lower sensitivity of 5-HT_2B receptors in mutants, with no change in 5-HT_2B mRNA. Blockade of 5-HT_2C receptors had no effect on NREMS in both strains. In addition, an increase in EEG power density after sleep deprivation was observed in 5-HT_2A+/+ mice but not in 5-HT_2A^-/- mice. Whole-body plethysmographic recordings indicated that 5-HT_2A receptor blockade in 5-HT_2A+/+ mice reduced NREMS apneas and bradypneas that occurred after sighs. In contrast, in 5-HT_2A^-/- mutants, NREMS apneas were not modified, and bradypnea after sighs were more pronounced. These results demonstrate that 5-HT exerts a 5-HT_2B-mediated facilitation of NREMS, and an influence respectively inhibitory on NREMS and facilitatory on sleep apnea generation, via 5-HT_2A receptors. Moreover, 5-HT_2A gene knock-out leads to functional compensations yielding adaptive changes opposite to those caused by pharmacological blockade of 5-HT_2A receptors in 5-HT_2A+/+ 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).

REFERENCES

Search PubMed for articles about Drosophila 5-hydroxytryptamine (serotonin) receptor 2A

Alex, K. D., et al. (2005). Modulation of dopamine release by striatal 5-HT2C receptors. Synapse 55(4): 242-51. 15668911

Berg, K. A., et al. (1998a). Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: evidence for agonist-directed trafficking of receptor stimulus. Mol. Pharmacol. 54(1): 94-104. PubMed Citation: 9658194

Berg, K. A., et al. (1998b). Pleiotropic behavior of 5-HT2A and 5-HT2C receptor agonists. Ann. N. Y. Acad. Sci. 861: 104-10. PubMed Citation: 9928246

Bhattacharyya, S., et al. (2006). Activation, internalization, and recycling of the serotonin 2A receptor by dopamine. Proc. Natl. Acad. Sci. 103(41): 15248-53. 17005723

Budnik, V., Wu, C.F. and White, K. (1989). Altered branching of serotonin-containing neurons in Drosophila mutants unable to synthesize serotonin and dopamine. J. Neurosci. 9: 2866-2877. PubMed Citation: 2570137

Burns, C. M., et al. (1997). Regulation of serotonin-2C receptor G-protein coupling by RNA editing. Nature 387(6630): 303-8. PubMed Citation: 9153397

Capela, J. P., et al. (2006). Ecstasy-induced cell death in cortical neuronal cultures is serotonin 2A-receptor-dependent and potentiated under hyperthermia. Neuroscience 139(3): 1069-81. 16504407

Chen, X., Reynolds, E., Ranganayakulu, G. and O'Donnell, J. (1994). A maternal product of the Punch locus of Drosophila melanogaster is required for precellular blastoderm nuclear divisions. J. Cell Sci. 107: 3501-3513. PubMed Citation: 7706401

Choi, D. S., et al. (1994a). The mouse 5-HT2B receptor: possible involvement in trophic functions of serotonin. Cell. Mol. Biol. (Noisy-le-grand) 40(3): 403-11. PubMed Citation: 7920184

Choi, D. S., Birraux, G., Launay, J.-M. and Maroteaux, L. (1994b). The human serotonin 5-HT2B receptor: pharmacological link between 5-HT2 and 5-HT1D receptors. FEBS Lett. 352, 393-399. PubMed Citation: 7926008

Choi, D. S. and Maroteaux, L. (1996a). Immunohistochemical localisation of the serotonin 5-HT2B receptor in mouse gut, cardiovascular system, and brain. FEBS Lett. 391(1-2): 45-51. PubMed Citation: 8706927

Choi, D. S., et al. (1996b). The mouse 5-HT2B receptor: homologous subtype or species variant? Behav. Brain Res. 73(1-2): 253-7. PubMed Citation: 8788513

Choi, D. S., et al. (1997). 5-HT2B receptor-mediated serotonin morphogenetic functions in mouse cranial neural crest and myocardiac cells. Development 124(9): 1745-55. 97307780

Choi, D. S., et al. (1998). Mouse 5-HT2B receptor-mediated serotonin trophic functions. Ann. N. Y. Acad. Sci. 861: 67-73

Colas, J.-F., Launay, J.-M., Kellermann, O., Rosay, P. and Maroteaux, L. (1995). Drosophila serotonin 5-HT2 receptor: Coexpression with fushi-tarazu during segmentation. Proc. Natl. Acad. Sci. 92: 5441-5445

Colas, J., Launay, J. and Maroteaux, L. (1999a). Maternal and zygotic control of serotonin biosynthesis are both necessary for Drosophila germband extension. Mech. Dev. 87(1-2): 67-76

Colas, J., et al. (1999b). Serotonin synchronises convergent extension of ectoderm with morphogenetic gastrulation movements in Drosophila. Mech. Dev. 87(1-2): 77-91

Cowen, D. S., Sowers, R. S. and Manning, D. R. (1996). Activation of a mitogen-activated protein kinase (ERK2) by the 5-hydroxytryptamine1A receptor is sensitive not only to inhibitors of phosphatidylinositol 3-kinase, but to an inhibitor of phosphatidylcholine hydrolysis. J. Biol. Chem. 271(37): 22297-300

Della Rocca, G. J., et al. (1999). Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis. J. Biol. Chem. 274(8): 4749-53

De Lucchini, S., et al. (2005). 5-HT2B-mediated serotonin signaling is required for eye morphogenesis in Xenopus. Mol. Cell. Neurosci. 29(2): 299-312. 15911353

Dickenson, J. M. and Hill, S. J. (1995). Coupling of an endogenous 5-HT1B-like receptor to increases in intracellular calcium through a pertussis toxin-sensitive mechanism in CHO-K1 cells. Br. J. Pharmacol. 116(7): 2889-96

Garnovskaya, M. N., et al. (1996). Ras-dependent activation of fibroblast mitogen-activated protein kinase by 5-HT1A receptor via a G protein beta gamma-subunit-initiated pathway. Biochemistry 35(43): 13716-22

Gerhardt, C. C., et al. (1996). Functional characterisation of a 5-HT2 receptor cDNA cloned from Lymnaea stagnalis. Eur. J. Pharmacol. 311(2-3): 249-58.

Goppelt-Struebe, M. and Stroebel, M. (1998). Signaling pathways mediating induction of the early response genes prostaglandin G/H synthase-2 and egr-1 by serotonin via 5-HT2A receptors. J. Cell Physiol. 75(3): 341-7

Guillet-Deniau, I., Burnol, A. F. and Girard, J. (1997). Identification and localization of a skeletal muscle secrotonin 5-HT2A receptor coupled to the Jak/STAT pathway. J. Biol. Chem. 272(23): 14825-9

Gunther, S., Maroteaux, L. and Schwarzacher, S. W. (2006). Endogenous 5-HT2B receptor activation regulates neonatal respiratory activity in vitro. J. Neurobiol. 66(9): 949-61. 16758492

Hagberg, G. B., et al. (1998). Stimulation of 5-HT2A receptors on astrocytes in primary culture opens voltage-independent Ca2+ channels. Neurochem. Int. 32(2): 153-62

Hamdan, F. F., et al. (1999). Characterization of a novel serotonin receptor from Caenorhabditis elegans: cloning and expression of two splice variants. J. Neurochem. 72(4): 1372-83

Hellendall, R. P., et al. (1993). Prenatal expression of 5-HT1C and 5-HT2 receptors in the rat central nervous system. Exp. Neurol. 120(2): 186-201

Hen, R. (1992). Of mice and flies: commonalities among 5-HT receptors. Trends Pharmacol. Sci. 13: 160-165

Horikawa, K., et al. (2000). Nonphotic entrainment by 5-HT_1A/7 receptor agonists accompanied by reduced Per1 and Per2 mRNA levels in the suprachiasmatic nuclei. J. Neurosci. 20(15): 5867-5873. 10908630

Hunt, P. N., McCabe, A. K., Gust, J. and Bosma, M. M. (2006a). Spatial restriction of spontaneous activity towards the rostral primary initiating zone during development of the embryonic mouse hindbrain. J. Neurobiol. 66(11): 1225-38. 16902989

Hunt, P. N., Gust, J., McCabe, A. K. and Bosma, M. M. (2006b). Primary role of the serotonergic midline system in synchronized spontaneous activity during development of the embryonic mouse hindbrain. J. Neurobiol. 66(11): 1239-52. 16902991

Irvine, K. and Wieschaus, E. (1994). Cell intercalation during Drosophila germband extension and its regulation by pairrule segmentation genes. Development 120: 827-841

Johnson, O., Becnel, J. and Nichols, C. D. (2011). Serotonin receptor activity is necessary for olfactory learning and memory in Drosophila melanogaster. Neuroscience 192: 372-81. PubMed Citation: 21749913

Kantor, S., Jakus, R., Balogh, B., Benko, A. and Bagdy, G. (2004). Increased wakefulness, motor activity and decreased theta activity after blockade of the 5-HT2B receptor by the subtype-selective antagonist SB-215505. Br. J. Pharmacol. 142(8): 1332-42. 15265808

Katayama, J., Yakushiji, T. and Akaike, N. (1997). Characterization of the K+ current mediated by 5-HT1A receptor in the acutely dissociated rat dorsal raphe neurons. Brain Res. 745(1-2): 283-92

Kerr, M., Davies, S. A. and Dow, J. A. T. (2004). Cell-specific manipulation of second messengers: A toolbox for integrative physiology in Drosophila. Curr. Biol. 14: 1468-1474. 15324663

Konrad, K. and Marsh, J. (1987). Developmental expression and spatial distri- bution of dopa decarboxylase in Drosophila. Dev. Biol. 122: 172-185

Launay, J.-M., Birraux, G., Bondoux, D., Callebert, J., Choi, D.-S., Loric, S. and Maroteaux, L. (1996). Ras involvement in signal transduction by the serotonin 5-HT2B receptor. J. Biol. Chem. 271, 3141-3147. 9621639

Lembo, P. M., et al. (1997). A conserved threonine residue in the second intracellular loop of the 5-hydroxytryptamine 1A receptor directs signaling specificity. Mol. Pharmacol. 52(1): 164-71

Lesurtel, M., et al. (2006). Platelet-derived serotonin mediates liver regeneration. Science 312(5770): 104-7. 16601191

Lin, H. W., Chen, C. C., Jhang, R. Y., Chen, L., de Belle, J. S., Tully, T. and Chiang, A. S. (2022). CREBB repression of protein synthesis in mushroom body gates long-term memory formation in Drosophila. Proc Natl Acad Sci U S A 119(50): e2211308119. PubMed ID: 36469774

Loric, S., Launay, J.-M., Colas, J.-F. and Maroteaux, L. (1992). New mouse 5-HT2-like receptor: Expression in brain, heart, and intestine. FEBS Lett. 312: 203-207

Loric, S., Maroteaux, L., Kellermann, O. and Launay, J.-M. (1995). Functional serotonin-2B receptors are expressed by a teratocarcinoma-derived cell line during serotoninergic differentiation. Mol. Pharmacol. 47: 458-466

Lucaites, V. L., et al. (1996). Receptor subtype and density determine the coupling repertoire of the 5-HT2 receptor subfamily. Life Sci. 59(13): 1081-95

McLean, J. R., Krishnakumar, S. and O'Donnell, J. M. (1993). Multiple mRNAs from the Punch locus of Drosophila melanogaster encode isoforms of GTP cyclohydrolase I with distinct N-terminal domains. J. Biol. Chem. 268: 27191-27197

Neckameyer, W. and White, K. (1992). A single locus encodes both phenylalanine hydroxylase and tryptophan hydroxylase activities in Drosophila. J. Biol. Chem. 267: 4199-4206

Ni, Y. G., Panicker, M. M. and Miledi, R. (1997). Efficient coupling of 5-HT1a receptors to the phospholipase C pathway in Xenopus oocytes. Brain Res. Mol. Brain Res. 51(1-2): 115-22

Nitsch, R. M., et al. (1996). Serotonin 5-HT2a and 5-HT2c receptors stimulate amyloid precursor protein ectodomain secretion. J. Biol. Chem. 271(8): 4188-94

Popa, D., et al. (2005). Contribution of 5-HT2 receptor subtypes to sleep-wakefulness and respiratory control, and functional adaptations in knock-out mice lacking 5-HT2A receptors. J. Neurosci. 25(49): 11231-8. 16339018

Reisoli, E., De Lucchini, S., Nardi, I. and Ori, M. (2010). Serotonin 2B receptor signaling is required for craniofacial morphogenesis and jaw joint formation in Xenopus. Development 137(17): 2927-37. PubMed Citation: 20667918

Reynolds, E. R. and O'Donnell, J. M. (1987). An analysis of the embryonic defects in Punch mutants of Drosophila melanogaster. Dev. Biol. 123: 430-441

Schmuck, K., et al. (1994). Cloning and functional characterization of the human 5-HT2B serotonin receptor. FEBS Lett. 342(1): 85-90

Sheffler. D. J., et al. (2006). p90 ribosomal S6 kinase 2 exerts a tonic brake on G protein-coupled receptor signaling. Proc. Natl. Acad. Sci. 103(12): 4717-22. 16537434

Sizemore, T. R. and Dacks, A. M. (2016). Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster. Sci Rep 6: 37119. PubMed ID: 27845422

Tournois, C., et al. (1998). Cross-talk between 5-hydroxytryptamine receptors in a serotonergic cell line. Involvement of arachidonic acid metabolism. J. Biol. Chem. 273(28): 17498-503

Walterscheid, J. P., et al. (2006). Cis-urocanic acid, a sunlight-induced immunosuppressive factor, activates immune suppression via the 5-HT2A receptor. Proc. Natl. Acad. Sci. 103(46): 17420-5. 17085585

Weisstaub, N. V., et al. (2006). Cortical 5-HT2A receptor signaling modulates anxiety-like behaviors in mice. Science 313(5786): 536-40. 16873667

Williams, C. L., Hayes, V. Y., Hummel, A. M., Tarara, J. E. and Halsey, T. J. (1993). Regulation of E-cadherin-mediated adhesion by muscarinic acet- ylcholine receptors in small cell lung carcinoma. J. Cell Biol. 121: 643-654

Wolf, W. A. and Schutz, L. J. (1997). The serotonin 5-HT2C receptor is a prominent serotonin receptor in basal ganglia: evidence from functional studies on serotonin-mediated phosphoinositide hydrolysis. J. Neurochem. 69(4): 1449-58

Wright, T.R.F. (1987). The genetics of biogenic amine metabolism, sclerotization, and melanization in Drosophila melanogaster. Adv. Genet. 24: 127-222

Wu, C., et al. (1999). Differential expression of serotonin 5-HT2 receptors during rat embryogenesis. Dev. Neurosci. 21(1): 22-8

Yavarone, M. S., et al. (1993). Serotonin and cardiac morphogenesis in the mouse embryo. Teratology 47(6): 573-84

Yin, J. C., Del Vecchio, M., Zhou, H. and Tully, T. (1995). CREB as a memory modulator: induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila. Cell 81(1): 107-115. PubMed ID: 7720066

Zhang, J., Tanenhaus, A. K., Davis, J. C., Hanlon, B. M. and Yin, J. C. (2015). Spatio-temporal in vivo recording of dCREB2 dynamics in Drosophila long-term memory processing. Neurobiol Learn Mem 118: 80-88. PubMed ID: 25460038

date revised: 25 October 2023

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