Gene name - Serotonin receptor 1A and Serotonin receptor 1B
Cytological map position-56B2-56B5 and 56B1-56B1
Function - transmembrane receptor
Symbol - 5-HT1A and 5-HT1B
Genetic map position - 2R
Classification - G-protein coupled receptor
Cellular location - surface
Although sleep is an important process essential for life, its regulation is poorly understood. The recently developed Drosophila model for sleep provides a powerful system to genetically and pharmacologically identify molecules that regulate sleep. Serotonin is an important neurotransmitter known to affect many behaviors, but its role in sleep remains controversial. Flies were generated with genetically altered expression of each of three Drosophila serotonin receptor subtypes (5-HT1A, 5-HT1B, and d5-HT2) and they were assayed for baseline sleep phenotypes. The data indicated a sleep-regulating role for the 5-HT1A receptor. 5-HT1A mutant flies had short and fragmented sleep, which was rescued by expressing the receptor in adult mushroom bodies, a structure associated with learning and memory in Drosophila. Neither the d5-HT2 receptor nor the 5-HT1B receptor, which was previously implicated in circadian regulation, had any effect on baseline sleep, indicating that serotonin affects sleep and circadian rhythms through distinct receptors. Elevating serotonin levels, either pharmacologically or genetically, enhanced sleep in wild-type flies. In addition, serotonin promoted sleep in some short-sleep mutants, suggesting that it can compensate for some sleep deficits. These data show that serotonin promotes baseline sleep in Drosophila. They also link the regulation of sleep behavior by serotonin to a specific receptor in a distinct region of the fly brain (Yuan, 2006).
Sleep is an essential part of animal physiology, with humans spending more than one-third of their lives in the sleep state. Sleep is also a complex process influenced by both genetic and environmental components. Despite the clear necessity for sleep and the extensive investigation of this process, the brain structures that drive sleep, the cellular mechanisms involved in sleep regulation, and the function of sleep are still unclear (Yuan, 2006).
Multiple approaches, including activity monitoring, arousal threshold measurements, rebound sleep following sleep deprivation, responsiveness to sleep-altering drugs, and electrophysiological studies, indicate that rest in Drosophila shares features with mammalian sleep. Studies in flies by both a candidate gene approach and large-scale genetic screens have identified several genes involved in sleep regulation, including the cAMP-dependent protein kinase (PKA), cAMP response element binding protein (CREB), a potassium channel, Shaker, and the dopamine transporter fumin (Hendricks, 2001; Cirelli, 2005; Kume, 2005). A study in mammals (Graves, 2003) confirmed a connection between CREB activity and the control of the sleep/wake cycle, suggesting that mechanisms of sleep regulation are conserved from flies to mammals (Yuan, 2006).
Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter widely distributed in the central and peripheral nervous systems of both mammals and insects. The idea that serotonin is involved in the regulation of sleep-wake cycles was proposed some time ago, but its role remains unclear. Reducing serotonin levels through pharmacological treatment or surgical ablation of serotonergic cells causes insomnia, suggesting that serotonin promotes sleep. In contrast, neuronal activity of serotonergic neurons and the timing of serotonin release suggest that it is associated with the waking state. Knockout mouse models for several serotonin receptor subtypes support an association between serotonin and sleep but have not clarified the mechanistic link. Overall, the results of many different approaches support the idea that serotonin suppresses rapid eye movement (REM) sleep; however, the effects on non-REM (NREM) sleep are debatable (Yuan, 2006 and references therein).
In Drosophila, serotonergic neurons send projections to most brain regions (Monastirioti, 1999; Lundell, 1994). Four serotonin receptors have been identified in the Drosophila genome, 5-HT1A, 5-HT1B, d5-HT2, and d5-HT7 (Saudou, 1992; Portas, 2000; Colas, 1995). They are all G protein-coupled receptors and share considerable sequence similarity with their mammalian homologs. Conserved effects of serotonin on the regulation of complex behaviors in flies and mammals were demonstrated in fly models of addiction, aggression, and circadian entrainment. This study considers the possibility that serotonin might affect sleep/arousal regulation in Drosophila. Therefore, the baseline sleep phenotypes of flies with genetically modified expression of three Drosophila serotonin receptors was tested. Flies carrying a truncated 5-HT1A receptor were shown to have decreased sleep amount and poor consolidation, while flies with reduced levels of the other two receptors, 5-HT1B and d5-HT2, show no baseline sleep abnormalities. The short and fragmented sleep phenotype of the 5-HT1A receptor mutant is rescued by a transgene of 5-HT1A expressed specifically in adult mushroom bodies. In addition, elevating serotonin levels pharmacologically or genetically promotes baseline sleep in wild-type flies. A serotonin precursor also enhances sleep in some known sleep mutants but fails to have effects when chemical neurotransmission is blocked in serotonergic cells, indicating that increased extracellular serotonin is required to promote sleep. It is proposed that the serotonin system is important for baseline sleep control in Drosophila and involves the function of the 5-HT1A receptor in adult mushroom bodies (Yuan, 2006).
This study utilized Drosophila as a model system to study the function of serotonin in sleep. The genetic tools available in Drosophila allowed study of loss-of-function mutants of three serotonin receptors: 5-HT1A, 5-HT1B, and d5-HT2. A significant effect on sleep was observed in flies with a truncated 5-HT1A receptor. These flies had short and fragmented sleep, which was rescued by expressing a 5-HT1A transgene in adult mushroom bodies. Pharmacological studies with a serotonin synthesis precursor achieved a similar effect in flies as in mammals, i.e., the promotion of sleep. These studies provide evidence for serotonergic signaling in the regulation of Drosophila sleep, identify a receptor that specifically functions in this process, and suggest that two serotonin receptor subtypes, 5-HT1A and 5-HT1B, mediate effects of serotonin on different behaviors in distinct regions of the brain (Yuan, 2006).
The lesion in the 5-HT1A mutant also affects the neighboring gene CG15117, which codes for a protein of unknown function, possibly involved in carbohydrate metabolism. Since the sleep phenotype was rescued by expression of 5-HT1A alone, it is believed that the sleep abnormality of the mutant was caused solely by the disruption of 5-HT1A and not the neighboring gene. While the data are definitive with respect to a role for 5-HT1A in sleep, it is acknowledged that at this point a role for 5-HT1B and 5-HT2 cannot be completely excluded. The loss-of-function mutants tested for these receptors are still capable of producing small amounts of transcript, although, at least in the 5-HT1B mutant, this transcript expression is too low to conduct its circadian function (Yuan, 2005). In addition, based on the sleep-enhancing effect of 5-HTP treatment in 5-HT1A mutant flies, it is thought that other unidentified serotonin receptors function in sleep regulation (Yuan, 2006).
Although they showed no obvious defects, homozygous flies carrying the truncated 5-HT1A receptor were less viable than flies with a single copy of the mutation in the same background. When maintained as heterozygotes, less than 5% of the progeny were homozygous for the 5-HT1A mutation. In homozygous stocks, the short-sleep phenotype weakened, such that sleep increased after several generations. This is consistent with the notion that the sleep phenotype is highly modifiable, especially when it is associated with reduced viability. A similar situation was reported for the Shaker mutants where the short-sleep phenotype diminished significantly within some generations in a w1118 background (Cirelli, 2005). For analysis of the 5-HT1A mutant, this problem was avoided by constantly maintaining a heterozygous stock and by employing a homozygous stock for no more than the first two generations. It is noted, however, that the 5-HT1A mutation was introduced into backgrounds containing different UAS or Gal4 transgenes for the rescue experiment, and it consistently maintained its phenotype. Thus, the effects of modifiers/background do not constitute an insurmountable problem in the analysis of sleep mutants (Yuan, 2006).
It is speculated that serotonin has a conserved function in sleep regulation. In mammals, systemic administration of 5-HTP or of an agonist of the mammalian 5-HT1A receptor increases slow-wave sleep (SWS) and reduces sleep latency. However, analysis of the mammalian 5-HT1A receptor has been complicated, in part because of the difficulty of dissociating its pre- and postsynaptic actions and also because of compensatory effects involving upregulation of other receptor subtypes. This may account for the lack of a NREM or total sleep phenotype in the 5-HT1A knockout mice. A sleep phenotype was identified in the Drosophila 5-HT1A mutant and an additional role for this receptor in the maintenance of sleep stability was demonstrated. It is thought that the phenotype in Drosophila results from the simpler structure of the fly genome with its less compensated serotonergic system. Also, while sleep in rodents is quite fragmented, flies, like humans, have prolonged sleep episodes. This may facilitate the identification of sleep-consolidating factors such as 5-HT1A (Yuan, 2006).
Associating the effect of serotonin on sleep with a specific receptor subtype provides a means to dissect the underlying circuitry and signaling pathways. Through genetic rescue experiments, it was found that 5-HT1A is required in the mushroom bodies to promote sleep stability. Previous studies demonstrated a role for 5-HT1B in circadian entrainment in clock cells and showed that this receptor is also expressed in the mushroom bodies (Yuan, 2005). However, 5-HT1B could not substitute for the function of 5-HT1A in fly sleep in rescue experiments, suggesting that, in addition to tissue-specific regulation, different downstream signaling is involved in fly behaviors modulated by serotonin. Although the two receptors share more than 80% homology in overall protein sequence, their N termini and third intracellular loops are very different, which could account for their molecular differences. In addition, it is possible that the expression of 1A and 1B in mushroom bodies is regulated either developmentally or spatially. These observations demonstrate that it is possible to link the regulatory effect of serotonin on complex behaviors in Drosophila with distinct receptor types and with specific neuronal structures (Yuan, 2006).
The localization of 5-HT1A action to the adult mushroom bodies is consistent with recent studies that indicate a sleep-regulating role for mushroom bodies (Joiner, 2006). The mushroom body is an important neuronal structure for control of locomotion and integration of sensory inputs, as well as learning and memory in flies. This study supports the association between sleep and memory consolidation. It is also consistent with the role of serotonin in learning and memory (Yuan, 2006).
A link between serotonin and human sleep has been demonstrated in clinical studies. Pathological conditions associated with serotonin deficits are linked to reduced sleep amount and quality. For instance, patients with clinical depression usually have sleep disorders. Antidepressant treatment upregulating serotonergic signaling improves their mental condition as well as their sleep quality. Results of these studies that used pharmacological and genetic approaches suggest that serotonin also promotes sleep in flies. In addition, these studies support the complementary conclusion, that is, flies with defects in serotonin signaling sleep less. Together, these results provide a model for studying the underlying mechanism and neuronal structures involved in the effect of serotonin on sleep (Yuan, 2006).
This study has shown that serotonin and the 5-HT1A receptor promote sleep in Drosophila. Serotonin can even increase sleep and sleep consolidation in some short-sleep mutants, suggesting that it has a central role in the control of sleep. The finding that 5-HT1A acts in the adult mushroom bodies to regulate sleep links sleep and serotonin signaling to learning and memory. Finally, given that effects of serotonin on circadian rhythms are mediated by the 5-HT1B receptor, and not by 5-HT1A, these data indicate nonoverlapping roles for serotonin receptor subtypes in the circadian and homeostatic control of sleep (Yuan, 2006).
The role of the mammalian 5-HT1A receptor in many animal behaviors, including aggression and sleep, has been demonstrated through pharmacological treatments and knockout mouse models (Boutrel, 2002; Ramboz, 1998). However, the involvement of this receptor in circadian regulation is supported only by pharmacological studies and thus is still uncertain (Smart, 2001). Of the four serotonin receptors identified in the Drosophila genome, the closest ortholog of the mammalian 5-HT1A receptor is the Drosophila serotonin receptor subtype 1B (5-HT1B), with 43% sequence similarity between the two receptors (Saudou, 1992). 5-HT1A and 5-HT1B are closely linked on chromosome 2R. The properties of 5-HT1B were analyzed in an S2 cell culture system. In response to serotonin and to 8-OH-DPAT, a mammalian 5-HT1A receptor specific agonist, the 5-HT1B receptor was internalized in intracellular vesicles and activated downstream mitogen-activated protein kinase (MAPK). Thus, this receptor shares molecular and pharmacological properties with its mammalian counterpart (Yuan, 2005).
date revised: 12 February 2007
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