One major piece of evidence supporting a sleep-promoting effect of serotonin in mammals is the effect of pharmacological agents that modulate serotonin levels. When delivered systemically, the serotonin synthesis inhibitor parachlorophenylalanine (pCPA) causes insomnia, which can be rescued by treatment with the serotonin synthesis precursor 5-hydroxytryptophan (5-HTP) (Jouvet, 1968). Previous reports suggested that systemic administration of pCPA does not deplete serotonin in the fly brain (Coleman, 2005). However, 5-HTP treatment increases serotonin levels in the CNS of both mammals and insects (Denoyer, 1989). To test whether increasing serotonin levels affects fly sleep, behavioral responses were assayed to chronic treatment with 5-HTP in female wild-type Canton-S flies. Doses of 5-HTP from 1 mg/ml to 5 mg/ml generated similar behavioral responses in Canton-S flies while the response to lower doses varied from fly to fly. Therefore, a 1 mg/ml dose was used in the following experiments. As compared to control flies, flies treated with 5-HTP had significantly increased amounts of sleep, which was more pronounced during the day. Because daytime sleep tends to be poorly consolidated, the increased sleep was manifested more as an increase in bout number than bout length. The lack of a significant effect upon nighttime sleep was most likely due to a “ceiling” effect in light of the fact that Canton-S female flies have high nighttime sleep. It was also observed that 5-HTP treatment reduced locomotor activity in flies. However, these effects of serotonin on fly locomotion were generally variable and could be dissociated from the effect of serotonin on sleep (Yuan, 2006).
To verify that the effect of 5-HTP on fly sleep was mediated through an increase in levels of extracelluar serotonin, the response to 5-HTP was tested in flies in which chemical neurotransmission was blocked in serotonergic cells. Flies carrying a UAS-TNT (tetanus neurotoxin light chain) transgene were crossed either to a Ddc-Gal4 driver, which drives expression in dopamine- and serotonin-producing cells, or to a TH-Gal4 driver, which is expressed in dopaminergic cells only. Progeny of each cross were tested in sleep assays with or without 5-HTP treatment and compared to their parental controls. Although daily sleep amount varied among genotypes, all parental control lines responded to the 5-HTP treatment with elevated daily sleep. However, baseline sleep in Ddc-Gal4/UAS-TNT flies did not change significantly in response to 5-HTP treatment. Further statistical analysis that used ANOVA to test for an interaction between genotype and drug treatment confirmed that the expression of UAS-TNT driven by Ddc-Gal4 had a significant effect on the sleep phenotype produced by 5-HTP. In contrast, TH-Gal4/UAS-TNT flies had elevated sleep, which increased further in the 5-HTP-treated group. The high baseline sleep phenotype in TH-Gal4/UAS-TNT flies is consistent with published data on the effect of the dopamine system in promoting arousal in flies (Kume, 2005; Andreic, 2005). At the same time, chemical silencing of the dopaminergic cells did not affect the response to 5-HTP treatment, indicating that the lack of a response to 5-HTP in Ddc-Gal4/UAS-TNT flies is due to the loss of serotonergic transmission (Yuan, 2006).
Next it was determined whether this effect of 5-HTP was universal across fly lines, in particular in lines with short-sleep phenotypes. All control strains tested, including yw, w1118, and Oregon-R, showed increased sleep in response to 5-HTP treatment. Interestingly, 5-HT1A mutants also exhibited a significant increase in sleep after treatment with 5-HTP, suggesting that increased serotonin levels may compensate for the deficit in 5-HT1A signaling, possibly through activating other unidentified serotonin receptors. Of the short-sleep mutants tested, flies carrying a loss-of-function mutation in a clock gene (cycle) or expressing a constitutively active protein kinase A molecule under the control of a leaky heat-shock promoter (Hendricks, 2001 and Hendricks, 2003) showed increased sleep in response to 5-HTP treatment. Since the mutants had decreased sleep at all times, sleep-promoting effects of 5-HTP were visible at night as well as during the day and, in fact, even increased nighttime sleep bout duration was observed. However, effects on nighttime sleep bout number were different in the two genotypes. Short-sleep flies mutant for a potassium channel (Shaker) (Cirelli, 2005; Kume, 2005) slept more in response to 5-HTP, but did not show increased bout duration at night. As a matter of fact, the Shaker flies showed reduced consolidation at night by both measures, but particularly in the greatly increased sleep bout number. Together, these data suggest that elevated serotonin can counteract the effects of other molecules that affect sleep, although to varying extents. It is speculated that the differences in the response of the mutants arise from differences in the mechanism of action of each of the respective mutations (Yuan, 2006).
To further investigate the effect of elevated serotonin on fly sleep, transgenic flies were generated with increased serotonin production. This was achieved by overexpressing an enzyme, tryptophan hydroxylase (TPH), which is responsible for catalyzing the conversion of tryptophan to 5-hydroxytryptamine, the first and the rate-limiting step in serotonin synthesis. As in the mammalian system, there are two TPH isoforms in the Drosophila genome, DTPH (henna) and DTRH (CG9122). The expression pattern of these two genes, as determined by in situ hybridization at a late embryonic stage, indicated that DTPH is expressed in the fat body, while DTRH is expressed in the central nervous system. Thus, DTRH is most likely the TPH isoform that is important for serotonin production in the Drosophila CNS (Yuan, 2006).
Overexpression of DTRH was driven by Ddc-Gal4 in serotonergic and dopaminergic cells, and the effect on serotonin levels was confirmed by ELISA assays of fly head extracts. Female flies with elevated serotonin levels were subjected to sleep analysis. As compared to control flies, Ddc-Gal4/UAS-DTRH flies had significantly increased total sleep and night sleep bout length, as well as reduced sleep bout number. Therefore, elevating serotonin levels through both pharmacological and genetic approaches enhances fly sleep (Yuan, 2006).
The enzymatic activity of Ebony regulates fly pigmentation, photoreceptor activity and behavioral rhythmicity. It has been suggested that glia may be required for normal circadian behavior, but glial factors required for rhythmicity have not been identified in any system. This study shows that a circadian rhythm in Drosophila Ebony (N-β-alanyl-biogenic amine synthetase) abundance can be visualized in adult glia and that glial expression of Ebony rescues the altered circadian behavior of ebony mutants. Molecular oscillator function and clock neuron output are normal in ebony mutants, verifying a role for Ebony downstream of the clock. Surprisingly, the ebony oscillation persists in flies lacking PDF neuropeptide, indicating it is regulated by an autonomous glial oscillator or another neuronal factor. The proximity of Ebony-containing glia to aminergic neurons and genetic interaction results suggest a function in dopaminergic signaling. A model for ebony function is presented wherein Ebony glia participate in the clock control of dopaminergic function and the orchestration of circadian activity rhythms (Suh, 2007).
Ebony plays a role in behavioral rhythmicity. Because life has evolved in the presence of daily geophysical cycles, most organisms have acquired the ability to adapt the timing of physiological processes to external cycles using an intrinsic time-keeping device called a circadian clock. Both forward genetic and molecular screens in Drosophila and other organisms have identified genes encoding integral components of the circadian oscillator. In the fruit fly, the core oscillator mechanism governing behavioral rhythmicity is comprised of two interconnected molecular loops that result in circadian changes in PER and TIM clock protein abundance and the cyclical feedback repression of clock gene transcription. In addition to the core transcriptional loops, posttranscriptional factors have been identified that are required for the modulation of clock protein stability, activity, or nuclear entry. Although there has been significant progress in delineating clock mechanisms, less is known about the molecular and cellular output pathways that control organismal physiology and behavior (Suh, 2007).
Two behaviors are widely employed to assay circadian rhythmicity in Drosophila: eclosion (the emergence of the adult from the pupal case) and adult locomotor activity. Mutation of a clock element affects rhythms in both eclosion and activity, since the same or a molecularly similar clock regulates both behaviors. In contrast, several mutations have been reported to affect only one of these two behaviors; i.e., to have rhythm-specific effects on circadian behavior. These findings indicate that genetically separable output pathways mediate the circadian control of the two different processes. Mutations in ebony, for example, selectively perturb the locomotor activity rhythm, causing arrhythmicity, but have no effect on the adult eclosion rhythm. Such a rhythm-specific effect suggests that Ebony acts downstream of the clock mechanism to orchestrate the circadian control of locomotor activity (Suh, 2007).
Multiple microarray-based studies have identified Drosophila transcripts exhibiting rhythmic daily changes in abundance. These studies verified cycling for all of the known clock genes and, importantly, identified hundreds of other genes that show robust circadian changes in abundance within head tissues. Of note, ebony RNA was shown to exhibit robust circadian cycling in two independent studies. These results are consistent with the behavioral studies discussed above, which suggest that Ebony protein functions in a clock output pathway (Suh, 2007).
The most obvious phenotype of ebony mutants is defective sclerotization and cuticle pigmentation, although they also exhibit altered rhythms, vision (Hotta, 1969), and courtship behavior. Consistent with these phenotypes, Ebony protein can be detected in the hypodermis (which produces the cuticle), the visual system, and other brain regions. In the fly visual system, Ebony is localized exclusively to glia including neuropile and epithelial glia, and it is thought that Ebony functions in a metabolic pathway that may terminate the action of histamine, the photoreceptor cell neurotransmitter. Based on studies of the pigmentation phenotype of ebony mutants, it was shown that Ebony protein has β-alanyl-dopamine (DA) synthase (BAS) enzymatic activity, and consequently mutants are lacking N-β-alanyl-dopamine (NBAD) in peripheral and neural tissues (Perez, 2004) and have elevated levels of DA and β-alanine in both types of tissues (Hodgetts, 1973; Ramadan, 1993). Recently, it was reported that the Ebony enzyme has a broader substrate specificity than anticipated from previous studies: purified Ebony can conjugate β-alanine to several different biogenic amines, including DA, serotonin (5-HT), histamine, tyramine, and octopamine; hence, it is now considered a β-alanyl-biogenic amine synthase (Suh, 2007 and references therein).
It is known that DA, 5-HT, and other biogenic amines have neuromodulatory activity in Drosophila and other insects. Together with the behavioral defects of ebony mutants, these findings suggest a model for the circadian function of Ebony, in which clock output regulates Ebony (BAS) activity, and consequent changes in biogenic amine-related signaling within a specific group of neural cells of the fly brain. This study shows that Ebony-containing glia are localized close to clock cell projections, that there is a PER/TIM-dependent control of rhythmic ebony expression within a discrete population of glial cells, and that Ebony enzymatic activity is required within glia for the clock control of locomotor activity. Cellular and molecular analyses indicate that Ebony acts downstream of the clock to control locomotor activity and that Ebony-containing glia are positioned near DA and 5-HT neurons of the larval and adult brains, consistent with the idea that these glia are required for the modulation of aminergic functions. A genetic interaction between ebony1 and an allele of the fly dopamine transporter gene (dDAT) suggests that dopaminergic transmission has a role in rhythmicity in vivo. That glia may function in rhythmicity is consistent with a genetic mosaic study that implies a role for PER/TIM-containing glia in the regulation of activity rhythms (Suh, 2007).
Studies of Ebony indicate that glia have an essential role in the orchestration of circadian locomotor activity. It is of interest that previous studies in both mammals and insects have suggested that glia might be important for the control of rhythmic physiological events. Cultured cortical astroglia that express per-luciferase transgenes, for example, show circadian rhythms of bioluminescence that may depend on diffusible signals from neurons of the suprachiasmatic nuclei; these studies suggest that such glia contain autonomous oscillators that can be reset by environmental stimuli or by interactions with clock neurons. In Drosophila, previous investigations have shown that the clock proteins PER and TIM can be detected in neurons and glia of the optic lobes and protocerebrum, and PER protein abundance fluctuates according to a circadian rhythm in both cell types. Consistent with roles for PER in neurons and glia, genetic mosaic analysis has suggested that per expression in either cell type might be sufficient for rhythmicity (albeit weak rhythmicity with glial expression). The current results indicate that Ebony is localized to glia of at least two types: those containing PER and TIM and a second class in which clock protein expression is not detectable. In the first class of cells, it seems likely that rhythmic ebony expression is controlled by an intracellular PER/TIM-based oscillator. In the latter class, ebony expression is most likely regulated by direct or indirect interactions with clock cells (Suh, 2007).
It is now an accepted axiom that neuron-glia interactions are critical for neuronal development and function. In addition to serving support roles in the mature nervous system, glial cells influence the developmental specification of neurons, migration, myelination, synapse number, and synaptic transmission. In Drosophila, studies have provided a detailed understanding of glial cell development and revealed the transcriptional mechanisms underlying the differentiation of this class of neural cells. Previous studies have shown that glial cells function in the phagocytosis of neuronal debris during development and documented roles for glia in injury-induced neuronal degeneration. Of note, a glial-specific receptor known as Draper has been described that is part of a neuron-glia signaling mechanism mediating such injury-induced responses. Insect glia have also been implicated in neurotransmitter uptake/recycling, based on studies of GABA, acetylcholine, or glutamate uptake. Finally, studies of Drosophila repo mutants have demonstrated that glial support is important for neuronal survival in insects, similar to results obtained in mammals (Suh, 2007).
Perhaps more relevant for behavior, recent studies have shown that certain types of mammalian glia (astrocytes) can regulate the excitability of neurons through the regulated release of 'gliotransmitters' (glutamate, ATP, adenosine, cytokines, and growth factors), and it has become apparent that there are reciprocal neuron-glia signaling systems that regulate neuronal excitability. Although certain aspects of this dynamic communication system are beginning to be understood, clearly much remains to be learned about the specific factors that regulate neuron-glia communication. These studies have identified a glial-specific factor (Ebony) and a subpopulation of glia within the fly nervous system that function with clock neurons to regulate circadian activity rhythms. It seems likely that intercellular communication between the neuronal and glial elements of the fly circadian system is important for the temporal coordination of activity (Suh, 2007).
Does PDF release contribute to the control of rhythmic ebony expression? Colocalization studies, using antibodies for Ebony and PDF, show that greater than 80% of the Ebony-containing glia reside in close proximity to PDF neuronal somata or projections — this is the case for glia that reside in the lateral and dorsal protocerebrum and the optic medulla of the adult brain. Previous immunoelectron microscopy results show that certain PDF-containing varicosities are adjacent to glial cells of the optic medulla. Ebony-containing glia have been observed near varicosities of the PDF neuronal projections, which probably contain dense core vesicles (DCVs), and adjacent to other regions of the projections. Surprisingly, however, it appears that PDF is not essential for the regulation of the ebony rhythm. It is therefore postulated that communication between Ebony glia and clock neurons, if it occurs, is mediated by factors other than the PDF neuropeptide (Suh, 2007).
A transgene expressing an enzymatically dead form of Ebony does not provide behavioral rescue for ebony mutants; thus, BAS activity is essential for Ebony's circadian function. As indicated previously, BAS can conjugate β-alanine to many different aminergic neurotransmitters, including DA, 5-HT, histamine, octopamine, and tyramine. Interestingly, it has been demonstrated that Ebony glia are situated near histamine release sites of photoreceptor cells in the lamina, and it has been suggested that BAS activity conjugates histamine to β-alanine to terminate action of the transmitter (Richardt, 2002). This study has shown that Ebony-containing cells are in close proximity to dopaminergic and serotonergic neurons of the larval and adult brains, suggesting a role for BAS in terminating DA and 5-HT action. A genetic interaction between e1 and DATfmn, a DAT mutant, strongly suggests that Ebony has a role in dopaminergic signaling. The rhythmic production of Ebony (BAS) may result in a circadian modulation of DA action and in turn rhythmic regulation of locomotor activity. Alternatively, circadian changes in BAS activity may result in the rhythmic production and release of N-β-alanyl-dopamine (NBAD) with high levels of NBAD driving locomotor activity. Two lines of evidence support this idea: (1) NBAD is presumably highest during the day, the time of maximal activity; (2) the e1 mutation, a protein null, eliminates NBAD, and this mutation suppresses the hyperactivity of DATfmn flies even though the double mutant is predicted to have high synaptic levels of DA (Suh, 2007).
This study has shown that Ebony glial expression is regulated in a circadian manner and that the protein is required within glia for normal behavioral rhythmicity. The localization of Ebony-containing glia near clock cells and aminergic neurons suggests an explicit model for Ebony regulation and function in the circadian system. According to this model, ebony transcription is regulated either directly by a PER/TIM-dependent oscillator within glia (for those glia containing PER and TIM) or by the release of an unidentified output factor from clock neurons. Consequently, diurnal changes in ebony-encoded and glial-localized BAS activity lead to rhythms in the conjugation of biogenic amines to β-alanine and generation of NBAA product (NBAD in glia near DA neurons). Such a diurnal modulation of amine action may help shape the temporal organization of the daily bouts of locomotor activity. This model, of course, implies the existence of a glial amine transporter that mediates the uptake of synaptic amines into glia, although such a system has not yet been identified in Drosophila (Suh, 2007).
Furthermore, it is postulated that the production of NBAD, which is high during the subjective day, serves as a bioactive compound to drive locomotor activity during the daytime. The observation that e1 mutants exhibit selective daytime deficits in locomotor activity is consistent with this idea. According to this model, NBAD is released from glia and acts on dopaminergic or other neurons to regulate excitability and/or transmitter release. There is no evidence in the literature that β-alanyl-amine conjugates have bioactivity, but this is certainly a possibility given that many other glial compounds have such activity. Obviously, NBAD may not regulate locomotor activity, by itself, as it is presumably high throughout the day, given the profile of Ebony production, whereas locomotor activity is bimodal, with bouts occurring at dawn and dusk. An alternative model for the role of Ebony in the regulation of activity is that the unconjugated amine (i.e., DA) provides excitatory drive for behavior and that its modification by BAS activity decreases such excitation. However, such a model is not consistent with the presumption that NBAD levels are highest during the daytime, the time of maximal activity, nor with the observation that the DATfmn;e1 mutant, which probably has high DA levels, is not hyperactive (Suh, 2007).
Finally, it is known that Drosophila tyrosine hydroxylase (TH) RNA is transcribed according to a circadian rhythm, with high abundance occurring during the subjective day, and it is thus a good assumption that TH enzymatic activity and DA production is maximal during the day. Such a profile of DA production may explain why a high constitutive expression of Ebony (BAS) in glia can restore rhythmic behavior. Because TH production is presumably still rhythmic in ebony mutants, high NBAD levels would be expected to occur in such flies only during the daytime, thus permitting behavioral rhythmicity (Suh, 2007).
There is virtually no information about the expression pattern or the role of d5-HT1B in adult flies. Analysis of this receptor was initiated by determining its spatial expression pattern. Transgenic flies were generating containing a genomic fragment of the d5-HT1B upstream region fused to GAL4. The d5-HT1B-GAL4 driver was crossed to UAS-GFP transgenic flies, and the expression pattern of the d5-HT1B promoter was then visualized through fluorescence microscopy. The expression of d5-HT1B is initiated in the late embryonic stage and continues through all developmental stages, with abundant expression in both larval and adult central nervous systems. In the adult fly brain, d5-HT1B expression was observed in the mushroom body, the pars intercerebralis (PI) neurons, a subgroup of dorsal neurons, the LNvs, the optic lobes, and SE5HT-IR neurons. To avoid transactivation caused by insertion sites of the Gal4 driver, multiple transgenic lines carrying independent insertions of the Gal4 driver on different chromosomes were tested, and similar patterns of GFP expression were observed (Yuan, 2005).
To determine the relationship of d5-HT1B-expressing neurons to serotonergic neurons and clock neurons, d5-HT1B-Gal4/UAS-GFP fly brains were costained for serotonin and PDF expression. The colocalization of d5-HT1B and PDF in larval, as well as adult fly brains, indicates that the receptor is expressed in LNvs at both stages; in adults it is expressed in large and small LNvs. There was no significant overlap of d5-HT1B and serotonin expression in the midbrain and the optic lobes. However, the expression of d5-HT1B in the SE5HT-IR neurons suggests that it might function as an autoreceptor in these cells (Yuan, 2005).
To study the expression pattern of the d5-HT1B protein, a polyclonal antibody was generated against the third intracellular loop of the receptor. The antibody reacts specifically with a protein of molecular weight ~70 kDa (which matches the predicted size of the d5-HT1B protein) in fly head extracts. In adult brain whole mounts, immunostaining using this antibody generated signals in the mushroom bodies, LNvs, dorsal neurons, PI neurons, and optic lobes. The same structures were labeled in the d5-HT1B-Gal4/UAS-GFP flies, indicating good agreement between the two methods used to visualize receptor expression. The stronger signals obtained with the antibody in the large LNvs and the PI neurons, and the relatively weaker signals in the small LNvs and the mushroom bodies, may be indicative of altered receptor stability in these cells. Sections of adult heads did not indicate any expression of d5-HT1B in the eye (Yuan, 2005).
To test possible circadian regulation of the d5-HT1B receptor, the temporal expression pattern of the d5-HT1B transcript and protein was determined using RNase protection assays and Western blots, respectively. There was no significant circadian variation in RNA or protein levels of d5-HT1B in the presence of LD cycles or in DD. However, receptor levels were affected in clock mutants. Levels were upregulated in timeless (tim01) flies and downregulated in cycle (cyc0) flies, suggesting possible effects of the circadian system on d5-HT1B protein levels (Yuan, 2005).
Entrainment of the Drosophila circadian clock to light involves the light-induced degradation of the clock protein timeless (Tim). This entrainment mechanism is inhibited by serotonin, acting through the Drosophila serotonin receptor 1B (5-HT1B). 5-HT1B is expressed in clock neurons, and alterations of its levels affect molecular and behavioral responses of the clock to light. Effects of 5-HT1B are synergistic with a mutation in the circadian photoreceptor cryptochrome (Cry) and are mediated by Shaggy (Sgg), Drosophila glycogen synthase kinase 3beta (GSK3beta), which phosphorylates Tim. Levels of serotonin are decreased in flies maintained in extended constant darkness, suggesting that modulation of the clock by serotonin may vary under different environmental conditions. These data identify a molecular connection between serotonin signaling and the central clock component Tim and suggest a homeostatic mechanism for the regulation of circadian photosensitivity in Drosophila (Yuan, 2005).
In mammals, serotonergic agents induce phase shifts in the behavioral rhythm similar to those produced by nonphotic stimuli such as locomotor activity (Glass, 2003). To address a role for serotonin in entrainment in Drosophila, adult flies were subjected to acute treatment (10 min) with serotonin at different circadian times in constant darkness (DD). Under these conditions, no evidence was seen for a significant phase shift produced by serotonin treatment. Extended treatment (24 hr) with agents that increase the level of extracellular serotonin, such as the synthesis precursor 5-Hydroxy-L-tryptophan (5-HTP) and the reuptake inhibitor fluoxetine hydrochloride (Prozac), did not produce phase shifts either. However, these treatments had an effect on light-induced phase shifts. Circadian photosensitivity in wild-type flies was measured as the magnitude of the phase shift induced by a short light pulse in the late night. Flies were first entrained to a 12:12 light:dark (LD) cycle, then transferred to DD and pulsed on the first day of DD at circadian time (CT) 20, which is 8 hr into the subjective dark period. The shift in phase was determined by comparing daily activity offsets in pulsed flies and unpulsed controls prior to and after the light pulse. Light pulses of two different light intensities were used. The magnitude of the phase shift depends upon the dose of the light pulse, and so a larger shift was observed with the more intense pulse. 5-HTP, Prozac, and another reuptake inhibitor, citalopram, reduced light-induced phase shifts significantly, particularly in response to the high intensity light pulse, suggesting that increased serotonin levels lead to decreased light responses in flies (Yuan, 2005).
To identify possible sites of interaction between the serotonin and circadian systems in Drosophila, their relative distribution in the fly brain was determined. Pigment-dispersing factor (PDF) is a marker for the ventral lateral neurons (LNvs), which are a major component of the Drosophila central clock. Adult fly brains expressing green fluorescent protein (GFP) under control of the Pdf promoter were stained with an anti-serotonin antibody. Cell bodies and projections of serotonergic neurons were observed in distinct areas of the brain. These include a cluster located in the posterior lateral subesophageal ganglion (SE5HT-IR). Serotonergic neurons were also observed close to the cell bodies of the LNvs. Using an mCD8::GFP reporter, it was found that large and small LNvs (l-LNvs and s-LNvs) receive projections from Dopa Decarboxylase (Ddc)-expressing cells. Although Ddc is expressed in dopaminergic and serotonergic neurons, in general these observations are consistent with the reported close spatial association of serotonergic systems and clock cells in other organisms (Yuan, 2005).
To investigate the role of d5-HT1B in circadian photosensitivity, particularly in the inhibitory effect of serotonin, 5-HT1B expression was knocked down through RNA interference using a UAS-5-HT1B RNAi transgene. The RNAi construct was expressed using the 5-HT1B-GAL4 driver; this reduced levels of endogenous 5-HT1B by >70% and decreased levels of overexpressed 5-HT1B. To test the effects of reduced 5-HT1B levels on circadian light sensitivity, these flies were exposed to light pulses of different intensities. 5-HT1B knockdown flies showed significantly increased phase shifts as compared to control flies. To determine if 5-HT1B is required for the inhibitory effects of serotonin on photosensitivity, the knockdown flies were treated with 5-HTP and light-induced phase shifts were assayed. As reported above for wild-type flies, the light response of control flies was inhibited by 5-HTP treatment. However, the same treatment did not have a significant effect on the 5-HT1B knockdown flies. Thus, flies with reduced levels of 5-HT1B display enhanced light-induced phase shifts that are not inhibited by 5-HTP treatment (Yuan, 2005).
The specific role of clock cells in the effect of 5-HT1B knockdown were tested by using a tim-gal4 driver. Flies in which 5-HT1B was knocked down with the tim-Gal4 driver showed significantly increased light-induced phase shifts at low light intensity as compared to control flies. In addition, 5-HTP treatment did not inhibit the light response of these flies. Thus, the effect of d5-HT1B knockdown does appear to be mediated, at least in part, by clock cells, although the tim-Gal4 driver had less of an effect than the 5-HT1B-Gal4 driver (Yuan, 2005). Photosensitivity of the knockdown flies was measured by assaying them under constant dim light conditions (<10 lux), which typically produce long periods in wild-type flies. Eexpression of the RNAi transgene specifically in cells that normally express 5-HT1B resulted in circadian periods significantly longer than those of controls, suggesting increased circadian photosensitivity in flies with reduced 5-HT1B levels. Use of the elav-Gal4 driver produced similar results. Together, these experiments indicate that 5-HT1B is part of a mechanism for modulating circadian photosensitivity and that it mediates inhibitory effects of serotonin on Drosophila circadian light responses (Yuan, 2005).
Serotonin regulates the entrainment of circadian behavioral rhythms in Drosophila by affecting the molecular response to light. By modulating the expression of the 5-HT1B receptor in clock neurons, a role of this receptor subtype has been established in the regulation of Drosophila circadian photosensitivity. The data also demonstrate that the molecular connection between 5-HT1B signaling and the clock is GSK3β, which directly phosphorylates the central clock component Tim. It is proposed that serotonin signaling is a part of the homeostatic regulation that prevents dramatic fluctuations in the phase of the circadian clock. In addition, given the altered levels of serotonin in extended DD, it may confer selectivity on the response of the clock to light under different environmental conditions (Yuan, 2005).
The expression pattern of 5-HT1B, as determined by both UAS-Gal4 experiments and by immunostaining, provides some clues to its functions in Drosophila. Besides LNvs and SE5HT-IR neurons, major compartments of the fly brain that express the 5-HT1B receptor include the optic lobes, PI neurons, and mushroom bodies. Interestingly, expression in each of these locations is consistent with functions proposed for serotonin signaling in other organisms. In the housefly, the neuropil of the optic lobes undergoes daily structural changes regulated possibly by serotonin and PDF. PI neurons are neurosecretory cells that may also participate in the ocellar phototransduction pathway. The mushroom body is important for olfactory learning and memory in Drosophila. Therefore, in addition to its postsynaptic function in the LNvs, 5-HT1B may be involved in other aspects of physiology and behavior (Yuan, 2005).
The effect of 5-HT1B on Tim was especially pronounced in the small LNvs. One of the differences between the large and small LNvs is in the timing of nuclear entry, which is delayed in the small subgroup. If delayed nuclear entry accounts for the increased resistance of Tim to light in the small LNvs, it would suggest that 5-HT1B signaling largely affects cytoplasmic Tim (Yuan, 2005).
In addition to its effect on the light response, 5-HT1B overexpression influences free-running behavioral rhythms of cryb flies. It is speculated that this is due to the loss of synchrony among LNs. The mutual coupling of oscillators within an organism is important for the generation and synchronization of circadian rhythms, and serotonin is implicated in this process in some insects. Decreased synchrony may also result from the reduced photosensitivity produced by 5-HT1B overexpression. Interestingly, a significant number of glass, cryb double mutants, which lack CRY as well as all visual photoreceptors, are arrhythmic in DD (Yuan, 2005).
5-HT1B not only affects circadian photosensitivity when over- or under-expressed, it also appears to be the major receptor subtype required for the inhibitory effects of serotonin on entrainment. Notably, when 5-HT1B was knocked down with the RNAi transgene driven by tim-Gal4, the effect on photosensitivity was not as pronounced as with the 5-HT1B-Gal4 driver. This might be due to some background differences in flies carrying the tim-Gal4 transgene, or to nonspecific effects produced by expressing the RNAi construct in irrelevant cells. Also, the possibility that cells other than clock neurons participate in the regulation of light sensitivity via 5-HT1B cannot be excluded. However, clock cells clearly have a major role in this effect, in particular since the circadian response to serotonin is eliminated in the tim-Gal4/RNAi flies (Yuan, 2005).
Effects of serotonin on circadian photosensitivity have been demonstrated in other systems, but the underlying mechanisms were not identified. These studies in Drosophila address this issue by demonstrating an effect of 5-HT1B signaling on the posttranslational modification of Tim via Sgg. In 5-HT1B-overexpressing flies, Tim phosphorylation is reduced, and its stability is increased. In contrast, Sgg phosphorylation is increased (i.e., its activity is decreased) in response to elevated levels of 5-HT1B as well as in response to serotonin treatment. Consistent with this effect of 5-HT1B on Sgg, increased Sgg activity abolishes effects of 5-HT1B overexpression on circadian photosensitivity, while 5-HT1B attenuates the period shortening produced by excess Sgg activity. These reciprocal effects in genetic experiments strongly support the regulation of Sgg activity by 5-HT1B. Expression data indicate that Sgg is expressed predominantly in the cytoplasm. The regulation of cytoplasmic Sgg by 5-HT1B is predicted to affect the phosphorylation status of Tim mainly in the cytoplasm; Sgg-phosphorylated Tim is transported to the nucleus more effectively and is also a better substrate for light-induced degradation (Yuan, 2005).
5-HT1B alone does not significantly affect circadian period, suggesting that its effects on Sgg are limited. In this context, it is noted that, while sgg hypomorphs have a period of ~26 hr, flies hemizygous for the locus have wild-type periods. It is inferred that small (up to 50%) changes in Sgg activity do not alter circadian period but can affect circadian photosensitivity. A role for Sgg in circadian photosensitivity was previously suggested by Martinek (2001) who found that forms of Tim phosphorylated by Sgg were selectively degraded in response to light. In fact, phosphorylated Tim is known to be more sensitive to light. While Sgg appears to be the primary kinase that increases photic sensitivity of Tim, the actual process of light-induced Tim degradation involves the activity of a tyrosine kinase (Yuan, 2005).
These results provide a new mechanism for circadian regulation by a G protein-coupled signaling pathway. A role for GSK3β in the mammalian circadian system was recently reported (Iwahana, 2004). In addition, the mammalian 5-HT1A receptor affects phosphorylation of GSK3β in the mouse brain. It is possible that inhibition of GSK3β activity is a conserved mechanism in the regulation of circadian entrainment in mammals and insects (Yuan, 2005).
Slow dark adaptation has been described in Drosophila, whereby circadian sensitivity to light increases more than 10-fold over 3 days in DD. Increased light responsiveness during dark adaptation occurs in rodents, but the mechanism underlying these effects has not been addressed. Elevated responsiveness to light after prolonged exposure to darkness could be due either to a gain in sensitivity in the sensory system or to an increase in sensory output, which may be caused by a reduction in an inhibitory mechanism. In this study, lower serotonin levels were observed in flies maintained in DD. Given that serotonin signaling modulates circadian light sensitivity, it may be the reduction in this inhibitory mechanism that at least partially accounts for the enhanced light response in prolonged DD (Yuan, 2005).
It is proposed that serotonin signaling, which is itself upregulated by light, is a part of a homeostatic mechanism that regulates circadian light sensitivity. A recent study using human subjects also suggested that serotonin levels in the brain reflect the duration of prior light exposure. This change in serotonin levels with light may be relevant to the etiology and treatment of seasonal affective disorder (SAD), a mood disorder related to the reduced hours of sunlight in winter, particularly at northern latitudes. SAD patients respond to antidepression drug treatments, as well as to light therapy, both of which may produce an increase in serotonin. The interplay of serotonin, light, and the circadian system suggests a close relationship between circadian regulation and mental fitness (Yuan, 2005).
Serotonin modulates the entrainment of the circadian system. In contrast, the current results, and studies done in mammalian systems also, suggest circadian effects on serotonin signaling. (1) Based upon the differences seen in LD versus DD in the fly brain, the level of serotonin is affected by the environmental light cycle. (2) Receptor levels are modulated by circadian components, since 5-HT1B levels are altered in fly circadian mutants. In addition, serotonin release and receptor activity are regulated in a circadian fashion in mammals. Mutual regulation of the circadian and serotonin systems may be necessary to maintain the normal physiological functions of both systems (Yuan, 2005).
Loss-of-function mutants of the Drosophila homologs of the mammalian 5-HT1 and 5-HT2 classes of receptors, which have been implicated in sleep regulation were collected or generated. Baseline sleep phenotypes were studied under alternating light-dark conditions in 7- to 10-day-old female flies bearing mutations in the following genes: 5-HT1A, 5-HT1B (both of which are Drosophila homologs of the mammalian 5-HT1A receptor), and d5-HT2. Total daily sleep and sleep bout length were used to describe overall sleep and sleep consolidation (Yuan, 2006).
The 5-HT1B receptor functions in the circadian response to light (Yuan, 2005). To assay the effect of 5-HT1B on sleep, UAS-5-HT1B and UAS-5-HT1BRNAi transgenes were expressed in different cell types by means of a variety of drivers including elav-Gal4 for panneuronal expression, tim-Gal4 for expression in clock cells, Ddc-Gal4 for expression in serotonin- and dopamine-producing cells, and 5-HT1B-Gal4 for expression in locations that express endogenous receptor. The effects of the UAS-5-HT1B and UAS-5-HT1BRNAi transgenes on increasing or decreasing, respectively, the levels of 5-HT1B protein and on circadian photosensitivity have been shown previously (Yuan, 2005). Daily sleep was compared between flies overexpressing 5-HT1B and those that had 5-HT1B expression knocked down through RNAi. Regardless of the Gal4 driver used to express the UAS transgenes or of the level of 5-HT1B receptor in a given region of the brain, no significant differences were found in either the total amount of sleep or in sleep consolidation measured as the average length of sleep bouts. These data suggest that the 5-HT1B receptor is not involved in the regulation of baseline sleep in Drosophila (Yuan, 2006).
The mammalian 5-HT2 receptor is implicated in several psychopathological conditions in humans, including schizophrenia and eating disorder. Pharmacological studies in mammals suggest that serotonin acts on 5-HT2 receptors in the thalamus to produce an arousing effect. In contrast, mouse knockouts of 5-HT2A and 5-HT2C have mildly reduced NREM sleep. Analysis of the Drosophila homolog of the 5-HT2 receptor is limited (Colas, 1995; Blenau, 2001), in part due to the lack of loss-of-function mutants (Yuan, 2006).
A fly line was obtained from the Exelixis collection carrying a piggybac insertion in the third intron of the d5-HT2 gene. Due to the presence of dual splice donor sites in this P element, the d5-HT2 transcript is disrupted, producing a loss-of-function allele. Specifically, the expression of three exons downstream of the insertion, which encode essential intracellular and transmembrane domains, is affected. Sleep analysis indicated that neither total sleep nor sleep bout length was significantly different between flies with reduced d5-HT2 levels and controls, suggesting that this receptor subtype is not involved in the regulation of fly baseline sleep (Yuan, 2006).
The 5-HT1A receptor, like the 5-HT1B receptor, is a homolog of the mammalian 5-HT1A receptor. It shares more than 80% homology with 5-HT1B and is located in close cytogenetic proximity. To generate a loss-of-function mutant of 5-HT1A, a fly line was obtained with a P element insertion 2.5 kb downstream of its coding region. Crosses were carried out to excise this element and ~500 independent P excision lines were screened. One P excision event generated an imprecise excision deleting more than 5 kb of genomic sequence including the 3′ coding region of 5-HT1A. In a baseline sleep assay, flies carrying deletions in 5-HT1A showed significantly reduced sleep and sleep bout length as compared to control flies from a precise excision line that does not affect the 5-HT1A receptor (Yuan, 2006).
The analysis of flies with modified expression levels of three Drosophila serotonin receptor subtypes suggested that 5-HT1A is a specific receptor subtype that regulates baseline sleep in flies. Detailed molecular and behavioral studies focused on the 5-HT1A mutant flies were carried out in the following experiments (Yuan, 2006).
To confirm that a loss-of-function mutant of 5-HT1A was generated, the deletion in the P excision line was carried out by genomic PCR and RT-PCR. Although 5-HT1A transcripts were still expressed in these flies, the last two exons, which encode the sixth and seventh transmembrane domains, the C-terminal end, and a part of the third intracellular loop, were deleted. In addition, the transcript produced by the neighboring gene CG15117, which encodes a putative metabolic enzyme, was also affected (Yuan, 2006).
Since flies carrying the deletion were capable of producing a truncated form of the receptor, the full-length and truncated receptors were cloned and tested in an S2 cell culture system. The truncated receptor was expressed diffusely in the cytoplasm, in contrast to the full-length receptor that localized largely to the cell surface. In response to serotonin, the full-length receptor on the cell surface was internalized and formed clusters while the truncated receptor showed no changes in its cytoplasmic distribution. Thus, consistent with functional studies of other C-terminal truncated G protein-coupled receptors, the lesion in the 5-HT1A gene leads to altered subcellular localization and loss of the response to serotonin. It is surmised that flies carrying the truncated receptor are loss-of-function mutants of 5-HT1A (Yuan, 2006).
The 5-HT1A mutant did not have visible defects in body and brain development or in locomotion. In circadian behavioral assays, 5-HT1A mutant flies showed normal free-running rhythms, although the strength of the rhythm was reduced. It is inferred that the reduced rhythm strength was due to increased nighttime activity resulting from the decrease in sleep. Unlike flies with reduced levels of the 5-HT1B receptor (Yuan, 2005), the 5-HT1A mutants did not show increased circadian photosensitivity as measured by light-induced phase shifts in the late night, suggesting nonredundant functions of these two receptors in the circadian system. Consistent with the lack of a role in circadian rhythms, transcript levels of 5-HT1A did not vary at different times of day (Yuan, 2006).
A detailed sleep analysis was conducted of 5-HT1A mutant flies. Sleep during the light and dark periods of the day was considered separately for higher resolution. Precise excision lines that did not affect the 5-HT1A gene, as well as heterozygous flies carrying one copy of the deletion, were used as background controls. The circadian profile of activity and sleep indicated that the onset and offset of activity as well as the distribution of sleep were similar in mutant and control flies. However, the 5-HT1A mutant had significantly reduced total sleep and sleep bout length, as well as an increased number of sleep bouts, indicating that sleep was both reduced and fragmented. Note that significant changes were noticed only for nighttime sleep in females, since they have limited and poorly consolidated daytime sleep. Although these studies focused largely on females, the gender of choice for sleep studies in Drosophila, sleep was also examined in male 5-HT1A mutant flies. These also had reduced overall sleep relative to their controls. However, since sleep in Drosophila is gender dimorphic, with daytime sleep accounting for a significant proportion of sleep in males, the sleep phenotype in 5-HT1A males was evident during the day and night (Yuan, 2006).
Sleep was assayed in constant dark conditions, which typically decrease sleep consolidation. As expected, when flies were transferred from light-dark cycles to constant darkness conditions, total sleep and sleep bout length were reduced, while sleep bout number increased. At the same time, the 5-HT1A mutant showed significantly larger changes in these parameters in response to the absence of light-dark cycles, suggesting that mechanisms for maintaining sleep stability are impaired in these flies. This was also indicated by their reduced homeostatic response to sleep deprivation. The 5-HT1A mutant and its background control were deprived of sleep by subjecting them to mechanical sleep deprivation for 6 hr in the latter half of the night. Compared to the precise excision control line, 1A mutant flies had significantly reduced sleep rebound (Yuan, 2006).
To genetically map the phenotype to the lesion in the 5-HT1A gene, deficiency-complementation tests were first carried out with a line carrying a deficiency in the genomic region of 5-HT1A (Df7550). Flies carrying the 5-HT1A mutant over the deficiency Df7550 had a short and fragmented sleep phenotype similar to that of the 5-HT1A homozygous mutant. Attempts were made to rescue the mutant sleep phenotype by generating transgenic flies carrying a UAS-5-HT1A construct. The transgene was introduced into the mutant background and expressed under the control of different drivers. To describe the effect of transgene expression on the sleep phenotype in mutant flies, focus was placed on total sleep and nighttime sleep bout length and bout number, parameters that are affected in the female 5-HT1A mutant. When the UAS-5-HT1A transgene was expressed panneuronally, driven by an elav-Gal4 driver, sleep levels and consolidation were restored in 5-HT1A mutants: total sleep and nighttime sleep bout length increased while bout number decreased, suggesting that the lesion in 5-HT1A was responsible for the sleep phenotype (Yuan, 2006).
To identify specific brain regions important for the function of 5-HT1A, its expression pattern was determined through in situ RNA hybridization. In whole-mount adult fly brains, the signal from hybridized 5-HT1A transcripts was observed largely in the mushroom bodies. Based on this expression pattern, attempts were made to determine if the sleep phenotype could be rescued through targeted expression of 5-HT1A in adult mushroom bodies. Therefore, the UAS construct was expressed under the control of an RU486-inducible mushroom body Gal4 driver, MB-Switch. 5-HT1A mutant flies carrying the UAS transgene and the inducible Gal4 driver were subjected to sleep tests, with one group treated with RU486 (500 μm, 1% ethanol) for expressing the transgene and another group serving as control (1% ethanol only). Total daily sleep and nighttime sleep bout length were restored to wild-type levels in RU486-treated Gal4-driven transgenic animals but not in uninduced controls. These results indicate that the effect on sleep of defective 5-HT1A signaling can be rescued by restoring expression of the wild-type gene exclusively in adult mushroom bodies (Yuan, 2006).
Based upon the high degree of sequence homology and similar molecular properties reported previously (Saudou, 1992), it is conceivable that 5-HT1A and 5-HT1B act through similar signaling pathways in distinct brain regions and can substitute for each other when expressed in a functionally relevant location. However, despite the similarity, a 5-HT1B transgene expressed in adult mushroom bodies did not rescue the 5-HT1A mutant phenotype. Tests were performed for possible genetic interactions between these two receptors by assaying sleep in flies with reduced levels of both. Knocking down the expression of 5-HT1B, by the UAS-1BRNAi transgene, in a 1A heterozygous mutant background did not produce any baseline sleep phenotype. Together, these data indicate a specific role for 5-HT1A in fly sleep that is carried out in adult mushroom bodies (Yuan, 2006).
As a first step toward population and quantitative genetic analysis of neurotransmitter receptors in Drosophila melanogaster, this study describes the parameters of nucleotide variation in three serotonin receptors and their association with pupal heart rate. Thirteen kilobases of DNA including the complete coding regions of 5-HT1A, 5-HT1B, and 5-HT2 were sequenced in 216 highly inbred lines extracted from two North American populations in California and North Carolina. Nucleotide and amino acid polymorphism is in the normal range for Drosophila genes and proteins, and linkage disequilibrium decays rapidly such that haplotype blocks are typically only a few SNPs long. However, intron 1 of 5-HT1A consists of two haplotypes that are at significantly different frequencies in the two populations. Neither this region of the gene nor any of the common amino acid polymorphisms in the three loci associate with either heart rate or heart rate variability. A cluster of SNPs in intron 2 of 5-HT1A, including a triallelic site, do show a highly significant interaction between genotype, sex, and population. While it is likely that a combination of weak, complex selection pressures and population structure has helped shape variation in the serotonin receptors of Drosophila, much larger sampling strategies than are currently adopted in evolutionary genetics will be required to disentangle these effects (Nikoh, 2004; full text of article).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila 5-HT1A and 5-HT1B
Alexandre, C., et al. (2006). Early life blockade of 5-hydroxytryptamine 1A receptors normalizes sleep and depression-like behavior in adult knock-out mice lacking the serotonin transporter. J. Neurosci. 26(20): 5554-64. 16707806
Amargos-Bosch, M., et al. (2004). Co-expression and in vivo interaction of serotonin1A and serotonin2A receptors in pyramidal neurons of prefrontal cortex. Cereb. Cortex 14(3): 281-99. 14754868
Andretic, R., van Swinderen, B., Greenspan, R. J. (2005). Dopaminergic modulation of arousal in Drosophila. Curr. Biol. 15(13): 1165-75. 16005288
Bailey, S. J. and Toth, M. (2004). Variability in the benzodiazepine response of serotonin 5-HT1A receptor null mice displaying anxiety-like phenotype: evidence for genetic modifiers in the 5-HT-mediated regulation of GABAA receptors. J. Neurosci. 24(28): 6343-51. 15254090
Blenau, W. and Baumann, A. (2001). Molecular and pharmacological properties of insect biogenic amine receptors: lessons from Drosophila melanogaster and Apis mellifera. Arch. Insect Biochem. Physiol. 48(1): 13-38. 11519073
Boutrel, B., Franc, B., Hen, R., Hamon, M. and Adrien, J. (1999). Key role of 5-HT1B receptors in the regulation of paradoxical sleep as evidenced in 5-HT1B knock-out mice. J. Neurosci. 19(8): 3204-12. 10191333
Cai, X., Gu, Z., Zhong, P., Ren, Y. and Yan, Z. (2002). Serotonin 5-HT1A receptors regulate AMPA receptor channels through inhibiting Ca2+/calmodulin-dependent kinase II in prefrontal cortical pyramidal neurons. J. Biol. Chem. 277(39): 36553-62. 12149253
Cirelli, C., Bushey, D., Hill, S., Huber, R., Kreber, R., Ganetzky, B. and Tononi, G. (2005). Reduced sleep in Drosophila Shaker mutants. Nature 434(7037): 1087-92. 15858564
Colas, J. F., Launay, J. M., Kellermann, O., Rosay, P. and Maroteaux, L. (1995). Drosophila 5-HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation. Proc. Natl. Acad. Sci. 92(12): 5441-5. 7777527
Coleman, C. M. and Neckameyer, W. S. (2005). Serotonin synthesis by two distinct enzymes in Drosophila melanogaster. Arch. Insect Biochem. Physiol. 59(1): 12-31. 15822093
Denoyer, M., Kitahama, K., Sallanon, M., Touret, M. and Jouvet, M. (1989). 5-Hydroxytryptophan uptake and decarboxylating neurons in the cat hypothalamus. Neuroscience 31(1): 203-11. 2788831
Glass, J. D., Grossman, G. H., Farnbauch, L. and DiNardo, L. (2003). Midbrain raphe modulation of nonphotic circadian clock resetting and 5-HT release in the mammalian suprachiasmatic nucleus. J. Neurosci. 23(20): 7451-60. 12930783
Gordon, J. A., Lacefield, C. O., Kentros, C. G. and Hen, R. (2005). State-dependent alterations in hippocampal oscillations in serotonin 1A receptor-deficient mice. J. Neurosci. 25(28): 6509-19. 16014712
Graves, L. A., et al. (2003). Genetic evidence for a role of CREB in sustained cortical arousal. J. Neurophysiol. 90(2): 1152-9. 12711709
Heisler, L. K., et al. (1998). Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice. Proc. Natl. Acad. Sci. 95(25): 15049-54. 9844013
Hendricks, J. C., et al. (2001). A non-circadian role for cAMP signaling and CREB activity in Drosophila rest homeostasis. Nat. Neurosci. 4: 1108-1115. 11687816
Hendricks, J. C., et al. (2003). Gender dimorphism in the role of cycle (BMAL1) in rest, rest regulation, and longevity in Drosophila melanogaster. J. Biol. Rhythms 18(1): 12-25. 12568241
Joiner, W. J., Crocker, A., White, B. H. and Sehgal, A. (2006). Sleep in Drosophila is regulated by adult mushroom bodies. Nature 441(7094): 757-60. 16760980
Jouvet, M. (1968). Insomnia and decrease of cerebral 5-hydroxytryptamine after destruction of the raphe system in the cat. Adv. Pharmacol. 6(Pt B): 265-79. 5301639
Kume, K., Kume, S., Park, S. K., Hirsh, J. and Jackson, F. R. (2005). Dopamine is a regulator of arousal in the fruit fly. J. Neurosci. 25(32): 7377-84. 16093388
Lemonde, S., et al. (2003). Impaired repression at a 5-hydroxytryptamine 1A receptor gene polymorphism associated with major depression and suicide. J. Neurosci. 23(25): 8788-99. 14507979
Li, Q., et al. (2004). Medial hypothalamic 5-hydroxytryptamine (5-HT)1A receptors regulate neuroendocrine responses to stress and exploratory locomotor activity: application of recombinant adenovirus containing 5-HT1A sequences. J. Neurosci. 24(48): 10868-77. 15574737
Lundell, M. J. and Hirsh, J. (1994). Temporal and spatial development of serotonin and dopamine neurons in the Drosophila CNS. Dev. Biol. 165(2): 385-96. 7958407
Monaca, C., Boutrel, B., Hen, R., Hamon, M. and Adrien, J. (2003). 5-HT 1A/1B receptor-mediated effects of the selective serotonin reuptake inhibitor, citalopram, on sleep: studies in 5-HT 1A and 5-HT 1B knockout mice. Neuropsychopharmacology 28(5): 850-6. 12637954
Monastirioti M. (1999). Biogenic amine systems in the fruit fly Drosophila melanogaster. Microsc. Res. Tech. 45(2): 106-21. 10332728
Muraki, Y., et al. (2004). Serotonergic regulation of the orexin/hypocretin neurons through the 5-HT1A receptor. J. Neurosci. 24(32): 7159-66. 15306649
Nikoh, N., Duty, A. and Gibson, G. (2004). Effects of population structure and sex on association between serotonin receptors and Drosophila heart rate. Genetics 168(4): 1963-74. 15611167
Ou, X. M., et al. (2000). Novel dual repressor elements for neuronal cell-specific transcription of the rat 5-HT1A receptor gene. J. Biol. Chem. 275(11): 8161-8. 10713139
Ou, X. M., et al. (2003). Freud-1: A neuronal calcium-regulated repressor of the 5-HT1A receptor gene. J. Neurosci. 23(19): 7415-25. 12917378
Papoucheva, E., et al. (2004). The 5-hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein. J. Biol. Chem. 279(5): 3280-91. 14604995
Portas, C. M., Bjorvatn, B. and Ursin, R. (2000). Serotonin and the sleep/wake cycle: special emphasis on microdialysis studies. Prog. Neurobiol. 60(1): 13-35. 10622375
Ramboz, S., et al. (1998). Serotonin receptor 1A knockout: an animal model of anxiety-related disorder. Proc. Natl. Acad. Sci. 95(24): 14476-81. 9826725
Santana, N., et al. (2004). Expression of serotonin1A and serotonin2A receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex. Cereb. Cortex 14(10): 1100-9. 15115744
Sarnyai, Z., et al. (2000). Impaired hippocampal-dependent learning and functional abnormalities in the hippocampus in mice lacking serotonin(1A) receptors. Proc. Natl. Acad. Sci. 97(26): 14731-6. 11121072
Saudou, F., et al. (1992). A family of Drosophila serotonin receptors with distinct intracellular signalling properties and expression patterns. EMBO J. 11(1): 7-17. 1310937
Smart, C. M. and Biello, S. M. (2001). WAY-100635, a specific 5-HT1A antagonist, can increase the responsiveness of the mammalian circadian pacemaker to photic stimuli. Neurosci. Lett. 305(1): 33-6. 11356301
Suh, J. and Jackson, F. R. (2007). Drosophila Ebony activity is required in glia for the circadian regulation of locomotor activity. Neuron 55(3): 435-47. Medline abstract: 17678856
Yuan, Q., Lin, F., Zheng, X. and Sehgal, A. (2005). Serotonin modulates circadian entrainment in Drosophila. Neuron 47(1): 115-27. 15996552
Yuan, Q., Joiner, W. J. and Sehgal, A. (2006). A sleep-promoting role for the Drosophila serotonin receptor 1A. Curr. Biol. 16(11): 1051-62. 16753559
Yuen, E. Y., et al. (2005). Serotonin 5-HT1A receptors regulate NMDA receptor channels through a microtubule-dependent mechanism. J. Neurosci. 25(23): 5488-501. 15944377
date revised: 12 February 2007
Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.