InteractiveFly: GeneBrief

Serotonin receptor 1A and Serotonin receptor 1B: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Serotonin receptor 1A and Serotonin receptor 1B

Synonyms -

Cytological map position-56B2-56B5 and 56B1-56B1

Function - transmembrane receptor

Keywords - sleep, brain, learning

Symbol - 5-HT1A and 5-HT1B

FlyBase ID: FBgn0004168 and FBgn0263116

Genetic map position - 2R

Classification - G-protein coupled receptor

Cellular location - surface



NCBI links for 5-HT1A: Precomputed BLAST | EntrezGene

NCBI links for 5-HT1B: Precomputed BLAST | EntrezGene
Recent literature
Qi, Y., Huang, J., Li, M. Q., Wu, Y. S., Xia, R. Y. and Ye, G. Y. (2016). Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors. Elife 5 [Epub ahead of print]. PubMed ID: 26974346
Summary:
Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. This study reports that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability, however the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B-null Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution.
BIOLOGICAL OVERVIEW

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).

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).

Neuromodulators often act through diverse sets of receptors expressed by distinct network elements and in this manner, differentially affect specific features of network dynamics. Knowing which network elements express each receptor for a given neuromodulator provides a framework for making predictions about the mechanistic basis by which a neuromodulator alters network activity. This study provides an 'atlas' of 5-HTR expression within the AL of Drosophila, thus revealing network elements subject to the different effects of serotonergic modulation. In summary, different receptors are predominantly expressed by distinct neuronal populations. For example, the 5-HT2B is expressed by ORNs, while the 5-HT2A and 7 are expressed by cholinergic PNs. Additionally, each receptor was found to be expressed by diverse populations of LNs, with the exception the 5-HT1B. For instance, 5-HT1A is expressed by GABAergic and peptidergic (TKK and MIP) LNs, while 5-HT2A and 2B are not expressed by peptidergic LNs. However, the vPNs are the exception to the general observation that distinct neuronal classes differ from each other in the 5-HTRs and the implications of this are discussed below. Together, these results suggest that within the AL, 5-HT differentially modulates distinct populations of neurons that undertake specific tasks in olfactory processing (Sizemore, 2016).

A recurring theme of neuromodulation is that the expression of distinct receptor types by specific neural populations allows a single modulatory neuron to differentially affect individual coding features. For instance, GABAergic medium spiny neurons (MSNs) in the nucleus accumbens express either the D1 or D2 dopamine receptor allowing dopamine to have opposite effects on different MSNs via coupling to different Galpha subunits (reviewed in56). MSNs that differ in dopamine receptor expression also differ in their synaptic connectivity. Dopamine activates D1-expressing MSNs that directly inhibit dopaminergic neurons in the ventral tegmental area (VTA), and inhibits D2-expressing MSNs that inhibit GABAergic VTA interneurons thus inducing suppression of dopamine release. In this manner, a single neuromodulator differentially affects two populations of principal neurons via different receptors to generate coordinated network output. This principle also holds true for the effects of 5-HT within the olfactory bulb. For instance, 5-HT enhances presynaptic inhibition of olfactory sensory neurons by 5-HT2C-expressing juxtaglomerular cells57, while increasing excitatory drive to mitral/tufted cells and periglomerular cells via 5-HT2A-expressing external tufted cells. Similarly, distinct classes of AL neurons were observed to differ in their expression of 5-HTRs. For instance, ePNs express the 5-HT2A, 5-HT2B and 5-HT7 receptors, while peptidergic LNs predominantly express the 5-HT1A receptor. This suggests that the cumulative effect of 5-HT results from a combination of differential modulation across neuronal populations within the AL. Interestingly, although it was found that 5-HT2B is expressed by ORNs, previous reports found that 5-HT does not directly affect Drosophila ORNs. In this study, ORNs were stimulated using antennal nerve shock in which the antennae were removed in order to place the antennal nerve within a suction electrode. Thus, if 5-HT2B is localized to the ORN cell body, removal of the antennae would eliminate any effect of 5-HT on ORNs. In several insects, 5-HT within the antennal haemolymph modulates ORN odor-evoked responses. Therefore, it is plausible ORNs are modulated by a source of 5-HT other than the CSD neurons within the AL. Serotonergic modulation of LN activity has widespread, and sometimes odor specific, effects on olfactory processing. LNs allow ongoing activity across the AL to shape the activity of individual AL neurons, often in a glomerulus specific manner creating non-reciprocal relationships. It is fairly clear that 5-HT directly modulates LNs, although 5-HT almost certainly affects synaptic input to LNs. Serotonin modulates isolated Manduca sexta LNs in vitro and, consistent with the current results, a small population of GABAergic LNs in the AL of Manduca also express the 5-HT1A receptor. Furthermore, 5-HT has odor-dependent effects on PN odor-evoked activity, suggesting that odor specific sets of lateral interactions are modulated by 5-HT. Different populations of LNs were found to express different sets of 5-HT receptors, however LNs were categorized based on transmitter type, so it is possible that these categories could be even further sub-divided based on morphological type, synaptic connectivity or biophysical characteristics. Regardless, the results suggest that 5-HT modulates lateral interactions within the AL by selectively affecting LN populations that undertake different tasks. For instance, the TKKergic LNs that express the 5-HT1A receptor provide a form of gain control by presynaptically inhibiting ORNs32. The results suggest that 5-HT may affect TKK mediated gain control differently relative to processes undertaken by other LN populations. Furthermore, the expression of the TKK receptor by ORNs is regulated by hunger, allowing the effects of TKK to vary with behavioral state. It would be interesting to determine if the expression of 5-HTRs themselves also vary with behavioral state as a means of regulating neuromodulation within the olfactory system (Sizemore, 2016).

Although it was primarily found that individual populations of AL neurons chiefly expressed a single or perhaps two 5-HTR types, the vPNs appear to be an exception. As a population, the vPNs express all of the 5-HTRs and the vPNs that express each 5-HTR did not appear to differ in terms of the proportion of those neurons that were GABAergic or cholinergic (roughly 3:2). Unfortunately, the approach does not allow determination of the degree to which individual vPNs co-express 5-HTRs. However, it is estimated that there are ~51 vPNs and even if this is an underestimate, there is likely some overlap of receptor types as a large number of vPNs expressed the 5-HT1A, 1B, 2B and 7 receptors. It is possible that a single vPN expresses one 5-HTR in the AL and a different 5-HTR in the lateral horn. However, the current approach only allows identification of which neurons express a given 5-HTR, not where that receptor is expressed. The CSD neurons ramify throughout both ALs and both lateral horns, thus vPNs could have differential spatial expression of individual 5-HTRs. Individual neurons expressing multiple 5-HTRs has been demonstrated in several neural networks. For instance, pyramidal cells in prefrontal cortex express both the 5-HT1A and 5-HT2A7. This allows 5-HT to have opposing effects that differ in their time course in the same cell. In terms of the vPNs, the results suggest that the current understanding of the diversity of this neuron class is limited. The expression of receptors for different signaling molecules could potentially be a significant component to vPN diversity (Sizemore, 2016).

Neuromodulators are often released by a small number of neurons within a network, yet they can have extremely diverse effects depending upon patterns of receptor expression. For the most part, individual populations of AL neurons differed in the receptor types that they expressed. This suggests that 5-HT differentially acts on classes of neurons that undertake distinct tasks in olfactory processing. In the case of the vPNs, this differential modulation may be fairly complex due to the diversity within this neuronal class. The goal of this study was to establish a functional atlas of 5-HTR expression in the AL of Drosophila. This dataset therefore provides a mechanistic framework for the effects of 5-HT on olfactory processing in this network (Sizemore, 2016).


REGULATION

Elevating Serotonin levels in flies promotes sleep

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).

Ebony activity is required in glia for the circadian regulation of locomotor activity: Ebony regulates β-alanyl conjugation of dopamine and histamine, thus 'trapping' these biogenic amines preventing their further function

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 (see Tyramine β hydroxylase); 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).

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

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

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

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

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

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

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

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

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

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

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


DEVELOPMENTAL BIOLOGY

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).


EFFECTS OF MUTATION

Serotonin modulates circadian entrainment in Drosophila

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).

Baseline sleep phenotypes in flies with modified levels of Drosophila serotonin receptors

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).

Characterization of the lesion in 5-HT1A mutants

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).

Phenotypic analysis of 5-HT1A mutant flies

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).

Mapping of the sleep phenotype to the 5-HT1A locus

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).

Effects of population structure and sex on association between serotonin receptors and Drosophila heart rate

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).


EVOLUTIONARY HOMOLOGS

Transcriptional regulation of Seratonin receptors

The level of expression of the 5-HT1A receptor in the raphe and limbic systems is implicated in the etiology and treatment of major depression and anxiety disorders. The rat 5-HT1A receptor gene is regulated by a proximal TATA-driven promoter and by upstream repressors that inhibit gene expression. Deletion of a 71-base pair (bp) segment between 1590/1519 bp of the 5-HT1A receptor gene induced over 10-fold enhancement of transcriptional activity in both 5-HT1A receptor-expressing (RN46A raphe and SN48 septal) cells and receptor-negative (L6 myoblast and C6 glioma) cells. A 31-bp segment of the repressor was protected from DNase I digestion by RN46A or L6 nuclear extracts. Within the 31-bp segment, a single protein complex was present in receptor-expressing cells that bound a novel 14-bp DNA element; in receptor-negative cells, an additional complex bound an adjacent 12-bp sequence. In receptor-positive but not receptor-negative cells, mutation of the 14-bp element to eliminate protein binding abrogated repression to nearly the same extent as deletion of the 1590/1519 bp segment. Additional mutation of both 14-bp and 12-bp elements abolished protein binding and repressor activity in receptor-negative cells. Thus a single protein-DNA complex at the 14-bp element represses the 5-HT1A receptor gene in 5-HT1A receptor-positive neuronal cells, whereas adjacent DNA elements provide a dual repression mechanism in 5-HT1A receptor-negative cells (Ou, 2000).

Altered regulation of 5-HT1A receptors is implicated in mood disorders such as anxiety and major depression. To provide insight into its transcriptional regulation, a novel DNA element [14 bp 5'-repressor element (FRE)] of the 5-HT1A receptor gene has been identified that mediates repression in neuronal and non-neuronal cells. A DNA binding protein [five' repressor element under dual repression binding protein-1 (Freud-1)] is reported that binds to FRE to mediate repression of the 5-HT1A receptor or heterologous promoters. Freud-1 is evolutionarily conserved and contains two DM-14 basic repeats, a predicted helix-loop-helix DNA binding domain, and a protein kinase C conserved region 2 (C2)/calcium-dependent lipid binding (CalB) calcium/phospholipid binding domain. An intact CalB domain was required for Freud-1-mediated repression. In serotonergic raphe cells, overexpression of Freud-1 repressed the 5-HT1A promoter and decreased 5-HT1A receptor protein levels, whereas transfection of antisense to Freud-1 derepressed the 5-HT1A gene and increased 5-HT1A receptor protein expression. Calcium-dependent signaling blocked Freud-1-FRE binding and derepressed the 5-HT1A promoter. Treatment with inhibitors of calmodulin or CAM-dependent protein kinase reversed calcium-mediated inhibition of Freud-1. Freud-1 RNA and protein were present in raphe nuclei, hippocampus, cortex, and hypothalamus, and Freud-1 protein was colocalized with 5-HT1A receptors, suggesting its importance in regulating 5-HT1A receptors in vivo. Thus, Freud-1 represents a novel calcium-regulated repressor that negatively regulates basal 5-HT1A receptor expression in neurons and may play a role in the altered regulation of 5-HT1A receptors associated with anxiety or major depression (Ou, 2003).

Palmitoylation of serotonin receptors

In the present study, it was verified that the mouse 5-HT1A receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT1A receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT1A receptor acylation, the ability of acylation-deficient mutants to interact with heterotrimeric Gi protein and to modulate downstream effectors was analyzed. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with GGi protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alphaGi subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT1A receptor is critical for the enabling of GGi protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alphaGi-mediated signaling (Papoucheva, 2004).

Tissue specific expression os serotonin receptors

Serotonergic 5-HT1A and 5-HT2A receptors are abundantly expressed in prefrontal cortex (PFC) and are targets of atypical antipsychotic drugs. They mediate, respectively, inhibitory and excitatory actions of 5-HT. The transcripts for both receptors are largely (approximately 80%) colocalized in rat and mouse PFC, yet their quantitative distribution in pyramidal and GABAergic interneurons is unknown. Double in situ hybridization histochemistry was used to estimate the proportion of pyramidal and GABAergic neurons expressing these receptor transcripts in rat PFC. The number of GABAergic interneurons (expressing GAD mRNA) was a 22% of glutamatergic neurons (expressing vGluT1 mRNA, considered as putative pyramidal neurons). 5-HT2A receptor mRNA was present in a large percentage of pyramidal neurons (from 55% in prelimbic cortex to 88% in tenia tecta), except in layer VI, where it was localized only in 30% of those neurons. 5-HT2A receptor mRNA was present in approximately 25% of GAD-containing cells except in layer VI (10%). Likewise, approximately 60% of glutamatergic cells contained the 5-HT1A receptor transcript. Approximately 25% of GAD-expressing cells contained the 5-HT1A receptor mRNA. These data help to clarify the role of 5-HT in prefrontal circuits and shed new light to the cellular elements involved in the action of atypical antipsychotics (Santana, 2004).

Serotonin receptor deficiency, cognition, stress responses, locomotor activity, and anxiety-related behavior

The brain serotonin system is a powerful modulator of emotional processes and a target of medications used in the treatment of psychiatric disorders. To evaluate the contribution of serotonin 5-HT1A receptors to the regulation of these processes, gene-targeting technology was used to generate 5-HT1A receptor-mutant mice. These animals lack functional 5-HT1A receptors as indicated by receptor autoradiography and by resistance to the hypothermic effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Homozygous mutants display a consistent pattern of responses indicative of elevated anxiety levels in open-field, elevated-zero maze, and novel-object assays. Moreover, they exhibit antidepressant-like responses in a tail-suspension assay. These results indicate that the targeted disruption of the 5-HT1A receptor gene leads to heritable perturbations in the serotonergic regulation of emotional state. 5-HT1A receptor-null mutant mice have potential as a model for investigating mechanisms through which serotonergic systems modulate affective state and mediate the actions of psychiatric drugs (Heisler, 1998).

To investigate the contribution of individual serotonin receptors to mood control, homologous recombination was used to generate mice lacking specific serotonergic receptor subtypes. Mice without 5-HT1A receptors display decreased exploratory activity and increased fear of aversive environments (open or elevated spaces). 5-HT1A knockout mice also exhibited a decreased immobility in the forced swim test, an effect commonly associated with antidepressant treatment. Although 5-HT1A receptors are involved in controlling the activity of serotonergic neurons, 5-HT1A knockout mice had normal levels of 5-HT and 5-hydroxyindoleacetic acid, possibly because of an up-regulation of 5-HT1B autoreceptors. Heterozygote 5-HT1A mutants expressed approximately one-half of wild-type receptor density and displayed intermediate phenotypes in most behavioral tests. These results demonstrate that 5-HT1A receptors are involved in the modulation of exploratory and fear-related behaviors and suggest that reductions in 5-HT1A receptor density due to genetic defects or environmental stressors might result in heightened anxiety (Ramboz, 1998).

The hippocampus is a major limbic target of the brainstem serotonergic neurons that modulate fear, anxiety, and learning through postsynaptic 5-HT1A receptors. Because chronic stress selectively down-regulates the 5-HT1A receptors in the hippocampus, it was hypothesized that mice lacking these receptors may exhibit abnormalities reminiscent of symptoms of stress-related psychiatric disorders. In particular, a hippocampal deficit in the 5-HT1A receptor could contribute to the cognitive abnormalities often seen in these disorders. To test whether a deficit in 5-HT1A receptors impairs hippocampus-related functions, hippocampal-dependent learning and memory, synaptic plasticity in the hippocampus, and limbic neuronal excitability was studied in 5-HT1A-knockout (KO) mice. 5-HT1A-KO animals showed a deficit in hippocampal-dependent learning and memory tests, such as the hidden platform (spatial) version of the Morris water maze and the delayed version of the Y maze. The performance of KO mice was not impaired in nonhippocampal memory tasks such as the visible platform (nonspatial) version of the Morris water maze, the immediate version of the Y maze, and the spontaneous-alternation test of working memory. Furthermore, paired-pulse facilitation in the dentate gyrus of the hippocampus was impaired in 5-HT1A-KO mice. Finally, 5-HT1A-KO mice, as compared with wild-type animals, displayed higher limbic excitability manifested as lower seizure threshold and higher lethality in response to kainic acid administration. These results demonstrate that 5-HT1A receptors are required for maintaining normal hippocampal functions and implicate a role for the 5-HT1A receptor in hippocampal-related symptoms, such as cognitive disturbances, in stress-related disorders (Sarnyai, 2000).

Inhibition of serotonergic raphe neurons is mediated by somatodendritic 5-HT1A autoreceptors, which may be increased in depressed patients. This study reports an association of the C(-1019)G 5-HT1A promoter polymorphism with major depression and suicide in separate cohorts. In depressed patients, the homozygous G(-1019) allele was enriched twofold versus controls, and in completed suicide cases the G(-1019) allele was enriched fourfold. The C(-1019) allele was part of a 26 bp imperfect palindrome that bound transcription factors nuclear NUDR [nuclear deformed epidermal autoregulatory factor (DEAF-1)]/suppressin and Hairy/Enhancer-of-split-5 (Drosophila) (Hes5) to repress 5-HT1A or heterologous promoters, whereas the G(-1019) allele abolished repression by NUDR, but only partially impaired Hes5-mediated repression. Recombinant NUDR bound specifically to the 26 bp palindrome, and endogenous NUDR was present in the major protein-DNA complex from raphe nuclear extracts. Stable expression of NUDR in raphe cells reduced levels of endogenous 5-HT1A protein and binding. NUDR protein was colocalized with 5-HT1A receptors in serotonergic raphe cells, hippocampal and cortical neurons, and adult brain regions including raphe nuclei, indicating a role in regulating 5-HT1A autoreceptor expression. These data indicate that NUDR is a repressor of the 5-HT1A receptor in raphe cells the function of which is abrogated by a promoter polymorphism. A novel transcriptional model is suggested in which the G(-1019) allele derepresses 5-HT1A autoreceptor expression to reduce serotonergic neurotransmission, predisposing to depression and suicide (Lemonde, 2003).

Previous studies found that serotonin transporter (SERT) knock-out mice showed increased sensitivity to minor stress and increased anxiety-like behavior but reduced locomotor activity. These mice also showed decreased density of 5-HT1A receptors in the hypothalamus, amygdala, and dorsal raphe. To evaluate the contribution of hypothalamic 5-HT1A receptors to these phenotypes of SERT knock-out mice, two studies were conducted. Recombinant adenoviruses containing 5-HT1A sense and antisense sequences (Ad-1AP-sense and Ad-1AP-antisense) were used to manipulate 5-HT1A receptors in the hypothalamus. The expression of the 5-HT1A genes is controlled by the 5-HT1A promoter, so that they are only expressed in 5-HT1A receptor-containing cells. Injection of Ad-1AP-sense into the hypothalamus of SERT knock-out mice restored 5-HT1A receptors in the medial hypothalamus; this effect was accompanied by elimination of the exaggerated adrenocorticotropin responses to a saline injection (minor stress) and reduced locomotor activity but not by a change in increased exploratory anxiety-like behavior. To further confirm the observation in SERT-/- mice, Ad-1AP-antisense was injected into the hypothalamus of normal mice. The density and the function of 5-HT1A receptors in the medial hypothalamus were significantly reduced in Ad-1AP-antisense-treated mice. Compared with a control group, Ad-1A-antisense-treated mice showed a significant reduction in locomotor activity, but again no changes in exploratory anxiety-like behaviors, tested by elevated plus-maze and open-field tests. Thus, the present results demonstrate that medial hypothalamic 5-HT1A receptors regulate stress responses and locomotor activity but may not regulate exploratory anxiety-like behaviors (Li, 2004).

Mice lacking the serotonin 1A receptor (5-HT1AR) show increased levels of anxiety-related behavior across multiple tests and background strains. Tissue-specific rescue experiments, lesion studies, and neurophysiological findings all point toward the hippocampus as a potential mediator of the phenotype. Serotonin, acting through 5-HT1ARs, can suppress hippocampal theta-frequency oscillations, suggesting that theta oscillations might be increased in the knock-outs. To test this hypothesis, local field potential recordings were obtained from the hippocampus of awake, behaving knock-outs and wild-type littermates. The magnitude of theta oscillations was increased in the knock-outs, specifically in the anxiety-provoking elevated plus maze and not in a familiar environment or during rapid eye movement sleep. Theta power correlated with the fraction of time spent in the open arms, an anxiety-related behavioral variable. These results suggest a possible role for the hippocampus, and theta oscillations in particular, in the expression of anxiety in 5-HT1AR-deficient mice (Gordon, 2005).

Serotonin receptors, circadian rhythms, and sleep

The involvement of 5-HT1B receptors in the regulation of vigilance states was assessed by investigating the spontaneous sleep-waking cycles and the effects of 5-HT receptor ligands on sleep in knock-out (5-HT1B-/-) mice that do not express this receptor type. Both 5-HT1B-/- and wild-type 129/Sv mice exhibited a clear-cut diurnal sleep-wakefulness rhythm, but knock-out animals were characterized by higher amounts of paradoxical sleep and lower amounts of slow-wave sleep during the light phase and by a lack of paradoxical sleep rebound after deprivation. In wild-type mice, the 5-HT1B agonists CP 94253 and RU 24969 induced a dose-dependent reduction of paradoxical sleep during the 2-6 hr after injection, whereas the 5-HT1B/1D antagonist GR 127935 enhanced paradoxical sleep. In addition, pretreatment with GR 127935, but not with the 5-HT1A antagonist WAY 100635, prevented the effects of both 5-HT1B agonists. In contrast, none of the 5-HT1B receptor ligands, at the same doses as those used in wild-type mice, had any effect on sleep in 5-HT1B-/- mutants. Finally, the 5-HT1A agonist 8-OH-DPAT induced in both strains a reduction in the amount of paradoxical sleep. Altogether, these data indicate that 5-HT1B receptors participate in the regulation of paradoxical sleep in the mouse (Boutrel, 1999).

Mammalian circadian rhythms are synchronized daily to light-dark cycles in the environment. The suprachiasmatic nucleus (SCN) is the proposed site of the major circadian pacemaker. Daily entrainment is believed to be influenced by inputs to the SCN, one of these being the dense serotonergic (5-HT) projection from the raphe nuclei. WAY-100635 is a potent and selective 5-HT1A receptor antagonist. In this study, the effects of WAY-100635 on phase-shifts of the hamster circadian pacemaker to light were investigated. Phase-delays after a light pulse administered during the early subjective night (15 min at CT14) were observed to be significantly greater following pre-treatment with WAY-100635 compared to light pulse alone. However, pre-treatment with WAY-100635 had no effect on the magnitude of phase-shifts to light at CT18, late in the subjective night. Serotonin may influence the responsiveness of the circadian pacemaker to photic stimuli. Specifically, WAY-100635 administered at CT14 can augment phase-shifts to light (Smart, 2001).

Selective serotonin reuptake inhibitors (SSRIs) are extensively used for the treatment of depression. Aside from their antidepressant properties, they provoke a deficit in paradoxical sleep (PS) that is most probably mediated by the transporter blockade-induced increase in serotonin concentration in the extracellular space. Such an effect can be accounted for by the action of serotonin at various types of serotonergic receptors involved in PS regulation, among which the 5-HT1A and 5-HT1B types are the best candidates. According to this hypothesis, the effects were examined of citalopram, the most selective SSRI available to date, on sleep in the mouse after inactivation of 5-HT1A or 5-HT1B receptors, either by homologous recombination of their encoding genes, or pharmacological blockade with selective antagonists. For this purpose, sleep parameters of knockout mice that do not express these receptors and their wild-type counterparts were monitored during 8 h after injection of citalopram alone or in association with 5-HT1A or 5-HT5-HT1B receptor antagonists. Citalopram induced mainly a dose-dependent inhibition of PS during 2-6 h after injection, which was observed in wild-type and 5-HT5-HT1B-/- mice, but not in 5-HT1A-/- mutants. This PS inhibition was fully antagonized by pretreatment with the 5-HT1A antagonist WAY 100635, but only partially with the 5-HT5-HT1B antagonist GR 127935. These data indicate that the action of the SSRI citalopram on sleep in the mouse is essentially mediated by 5-HT1A receptors. Such a mechanism of action provides further support to the clinical strategy of antidepressant augmentation by 5-HT1A antagonists, because the latter would also counteract the direct sleep-inhibitory side-effects of SSRIs (Monaca, 2003).

In serotonin transporter knock-out (5-HTT-/-) mice, extracellular serotonin (5-HT) levels are markedly elevated in the brain, and rapid eye movement sleep (REMS) is enhanced compared with wild-type mice. It is hypothesized that such sleep impairment at adulthood results from excessive serotonergic tone during early life. Thus, tests were performed to see if neonatal treatment with drugs capable of limiting the impact of 5-HT on the brain could normalize sleep patterns in 5-HTT-/- mutants. It was found that treatments initiated at postnatal day 5 and continued for 2 weeks with the 5-HT synthesis inhibitor para-chlorophenylalanine, or for 4 weeks with the 5-HT1A receptor (5-HT1AR) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), induced total or partial recovery of REMS, respectively, in 5-HTT-/- mutants. Early life treatment with WAY 100635 also reversed the depression-like behavior otherwise observed in these mutants. Possible adaptive changes in 5-HT1AR after neonatal treatment with WAY 100635 were investigated by measuring 5-HT1A binding sites and 5-HT1A mRNA in various REMS- and/or depression-related brain areas, as well as 5-HT1AR-mediated hypothermia and inhibition of neuronal firing in the dorsal raphe nucleus. None of these characteristics were modified in parallel with REMS recovery, suggesting that 5-HT1ARs involved in wild-type phenotype rescue in 5-HTT-/- mutants are located in other brain areas or in 5-HT1AR-unrelated circuits where they could be transiently expressed during development. The reversal of sleep alterations and depression-like behavior after early life blockade of 5-HT1AR in 5-HTT-/- mutants might open new perspectives regarding preventive care of sleep and mood disorders resulting from serotonin transporter impairments during development (Alexandre, 2006).

Serotonin receptors regulate Glutamate receptor channels

The regulation of AMPA receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC) was studied. Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT1A receptor agonists and blocked by 5-HT1A antagonists, indicating the mediation by 5-HT1A receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT1A agonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. It is concluded that serotonin, by activating 5-HT1A receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice (Cai, 2002).

The serotonin system and NMDA receptors (NMDARs) in prefrontal cortex (PFC) are both critically involved in the regulation of cognition and emotion under normal and pathological conditions; however, the interactions between them are essentially unknown. Serotonin, by activating 5-HT1A receptors, inhibits NMDA receptor-mediated ionic and synaptic currents in PFC pyramidal neurons, and the NR2B subunit-containing NMDA receptor is the primary target of 5-HT1A receptors. This effect of 5-HT1A receptors is blocked by agents that interfere with microtubule assembly, as well as by cellular knock-down of the kinesin motor protein KIF17 (kinesin superfamily member 17), which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Inhibition of either CaMKII (calcium/calmodulin-dependent kinase II) or MEK/ERK (mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) abolished the 5-HT1A modulation of NMDAR currents. Biochemical evidence also indicates that 5-HT1A activation reduced microtubule stability, which was abolished by CaMKII or MEK inhibitors. Moreover, immunocytochemical studies show that 5-HT1A activation decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Together, these results suggest that serotonin suppresses NMDAR function through a mechanism dependent on microtubule/kinesin-based dendritic transport of NMDA receptors that is regulated by CaMKII and ERK signaling pathways. The 5-HT1A-NMDAR interaction provides a potential mechanism underlying the role of serotonin in controlling emotional and cognitive processes subserved by PFC (Yuen, 2005).

Serotonin mediated regulation of GABAA

Benzodiazepines (BZs) acting as modulators of GABAA receptors (GABAARs) are an important group of drugs for the treatment of anxiety disorders. However, a large inter-individual variation in BZ sensitivity occurs in the human population with some anxiety disorder patients exhibiting diminished sensitivity to BZ and reduced density of GABAARs. The mechanism underlying BZ treatment resistance is not known, and it is not possible to predict whether an anxiety patient will respond to BZ. 5-hydroxytryptamine1A receptor (5-HT1AR) null mice (R-/-) on the Swiss-Webster (SW) background reproduce several features of BZ-resistant anxiety; they exhibit anxiety-related behaviors, do not respond to BZ, have reduced BZ binding, and have decreased expression of the major GABAAR subunits alpha1 and alpha2. R-/- mice on the C57Bl6 (B6) background also have anxiety phenotype, but they respond to BZ and have normal GABAAR subunit expression. This indicates that the 5-HT1AR-mediated regulation of GABAAR alpha subunit expression is subject to genetic modification. Hybrid SW/B6-R-/- mice also exhibit BZ-resistant anxiety, suggesting that SW mice carry a genetic modifier, which mediates the effect of the 5-HT1AR on the expression of GABAARalpha subunits. In addition, this genetic interaction in SW mice operates early in postnatal life to influence the expression of GABAAR alpha subunits at the transcriptional level. These data indicate that BZ-resistant anxiety results from a developmental arrest of GABAAR expression in SW-R-/- mice, and a similar mechanism may be responsible for the BZ insensitivity of some anxiety patients (Bailey, 2004).

Effects of antipsychotic drugs acting through serotonin receptors

The prefrontal cortex plays a key role in the control of higher brain functions and is involved in the pathophysiology and treatment of schizophrenia. Approximately 60% of the neurons in rat and mouse prefrontal cortex express 5-HT1A and/or 5-HT2A receptor mRNAs, which are highly co-localized (approximately 80%). The electrical stimulation of the dorsal and median raphe nuclei elicited 5-HT1A-mediated inhibitions and 5-HT2A-mediated excitations in identified pyramidal neurons recorded extracellularly in rat medial prefrontal cortex (mPFC). Opposite responses in the same pyramidal neuron could be evoked by stimulating the raphe nuclei at different coordinates, suggesting a precise connectivity between 5-HT neuronal subgroups and 5-HT1A and 5-HT2A receptors in pyramidal neurons. Microdialysis experiments showed that the increase in local 5-HT release evoked by the activation of 5-HT2A receptors in mPFC by DOI (5-HT2A/2C receptor agonist) was reversed by co-perfusion of 5-HT1A agonists. This inhibitory effect was antagonized by WAY-100635 and the prior inactivation of 5-HT1A receptors in rats and was absent in mice lacking 5-HT1A receptors. These observations help to clarify the interactions between the mPFC and the raphe nuclei, two key areas in psychiatric illnesses and improve understanding of the action of atypical antipsychotics, acting through these 5-HT receptors (Amargos-Bosch, 2004).

Both orexin and serotonin (5-HT) have important roles in the regulation of sleep-wakefulness, as well as in feeding behavior. The effects of 5-HT on orexin/hypocretin neurons was examined, using hypothalamic slices prepared from orexin/enhanced green fluorescent protein (EGFP) transgenic mice in which EGFP is expressed exclusively in orexin neurons. Patch-clamp recording from EGFP-expressing cells showed that 5-HT hyperpolarized all orexin neurons in a concentration-dependent manner. The response was inhibited by the 5-HT1A receptor antagonist WAY100635. A 5-HT1A receptor agonist, 8-hydroxy-2-(dl-N-propyl-amino)tetralin, also evoked hyperpolarization on orexin neurons with potency comparable with 5-HT. A low concentration of Ba2+ (30 microM) inhibited 5-HT-induced hyperpolarization. Single-channel recording revealed that the conductance of 5-HT-induced channel activity was 33.8 pS, which is in good agreement with that of the G-protein-coupled inward rectifier potassium channel (GIRK). Moreover, 5-HT1A receptor-like immunoreactivity was observed on orexin neurons, and 5-HT transporter immunoreactive nerve endings are in close apposition to orexin neurons. Intracerebroventricular injection of the 5-HT1A receptor-selective antagonist WAY100635 increased locomotor activity during the latter half of dark phase in wild-type mice but not in orexin/ataxin-3 mice in which orexin neurons are specifically ablated, suggesting that activation of orexin neurons is necessary for the WAY100635-induced increase in locomotor activity. These results indicate that 5-HT hyperpolarizes orexin neurons through the 5-HT1A receptor and subsequent activation of the GIRK and that this inhibitory serotonergic input to the orexin neurons is likely to be important for the physiological regulation of this neuropeptide system (Muraki, 2004).

Transcriptional regulation of Seratonin receptors

The level of expression of the 5-HT1A receptor in the raphe and limbic systems is implicated in the etiology and treatment of major depression and anxiety disorders. The rat 5-HT1A receptor gene is regulated by a proximal TATA-driven promoter and by upstream repressors that inhibit gene expression. Deletion of a 71-base pair (bp) segment between 1590/1519 bp of the 5-HT1A receptor gene induced over 10-fold enhancement of transcriptional activity in both 5-HT1A receptor-expressing (RN46A raphe and SN48 septal) cells and receptor-negative (L6 myoblast and C6 glioma) cells. A 31-bp segment of the repressor was protected from DNase I digestion by RN46A or L6 nuclear extracts. Within the 31-bp segment, a single protein complex was present in receptor-expressing cells that bound a novel 14-bp DNA element; in receptor-negative cells, an additional complex bound an adjacent 12-bp sequence. In receptor-positive but not receptor-negative cells, mutation of the 14-bp element to eliminate protein binding abrogated repression to nearly the same extent as deletion of the 1590/1519 bp segment. Additional mutation of both 14-bp and 12-bp elements abolished protein binding and repressor activity in receptor-negative cells. Thus a single protein-DNA complex at the 14-bp element represses the 5-HT1A receptor gene in 5-HT1A receptor-positive neuronal cells, whereas adjacent DNA elements provide a dual repression mechanism in 5-HT1A receptor-negative cells (Ou, 2000).

Altered regulation of 5-HT1A receptors is implicated in mood disorders such as anxiety and major depression. To provide insight into its transcriptional regulation, a novel DNA element [14 bp 5'-repressor element (FRE)] of the 5-HT1A receptor gene has been identified that mediates repression in neuronal and non-neuronal cells. A DNA binding protein [five' repressor element under dual repression binding protein-1 (Freud-1)] is reported that binds to FRE to mediate repression of the 5-HT1A receptor or heterologous promoters. Freud-1 is evolutionarily conserved and contains two DM-14 basic repeats, a predicted helix-loop-helix DNA binding domain, and a protein kinase C conserved region 2 (C2)/calcium-dependent lipid binding (CalB) calcium/phospholipid binding domain. An intact CalB domain was required for Freud-1-mediated repression. In serotonergic raphe cells, overexpression of Freud-1 repressed the 5-HT1A promoter and decreased 5-HT1A receptor protein levels, whereas transfection of antisense to Freud-1 derepressed the 5-HT1A gene and increased 5-HT1A receptor protein expression. Calcium-dependent signaling blocked Freud-1-FRE binding and derepressed the 5-HT1A promoter. Treatment with inhibitors of calmodulin or CAM-dependent protein kinase reversed calcium-mediated inhibition of Freud-1. Freud-1 RNA and protein were present in raphe nuclei, hippocampus, cortex, and hypothalamus, and Freud-1 protein was colocalized with 5-HT1A receptors, suggesting its importance in regulating 5-HT1A receptors in vivo. Thus, Freud-1 represents a novel calcium-regulated repressor that negatively regulates basal 5-HT1A receptor expression in neurons and may play a role in the altered regulation of 5-HT1A receptors associated with anxiety or major depression (Ou, 2003).

Palmitoylation of serotonin receptors

In the present study, it was verified that the mouse 5-HT1A receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT1A receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT1A receptor acylation, the ability of acylation-deficient mutants to interact with heterotrimeric Gi protein and to modulate downstream effectors was analyzed. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with GGi protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alphaGi subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT1A receptor is critical for the enabling of GGi protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alphaGi-mediated signaling (Papoucheva, 2004).

Tissue specific expression os serotonin receptors

Serotonergic 5-HT1A and 5-HT2A receptors are abundantly expressed in prefrontal cortex (PFC) and are targets of atypical antipsychotic drugs. They mediate, respectively, inhibitory and excitatory actions of 5-HT. The transcripts for both receptors are largely (approximately 80%) colocalized in rat and mouse PFC, yet their quantitative distribution in pyramidal and GABAergic interneurons is unknown. Double in situ hybridization histochemistry was used to estimate the proportion of pyramidal and GABAergic neurons expressing these receptor transcripts in rat PFC. The number of GABAergic interneurons (expressing GAD mRNA) was a 22% of glutamatergic neurons (expressing vGluT1 mRNA, considered as putative pyramidal neurons). 5-HT2A receptor mRNA was present in a large percentage of pyramidal neurons (from 55% in prelimbic cortex to 88% in tenia tecta), except in layer VI, where it was localized only in 30% of those neurons. 5-HT2A receptor mRNA was present in approximately 25% of GAD-containing cells except in layer VI (10%). Likewise, approximately 60% of glutamatergic cells contained the 5-HT1A receptor transcript. Approximately 25% of GAD-expressing cells contained the 5-HT1A receptor mRNA. These data help to clarify the role of 5-HT in prefrontal circuits and shed new light to the cellular elements involved in the action of atypical antipsychotics (Santana, 2004).

Serotonin receptor deficiency, cognition, stress responses, locomotor activity, and anxiety-related behavior

The brain serotonin system is a powerful modulator of emotional processes and a target of medications used in the treatment of psychiatric disorders. To evaluate the contribution of serotonin 5-HT1A receptors to the regulation of these processes, gene-targeting technology was used to generate 5-HT1A receptor-mutant mice. These animals lack functional 5-HT1A receptors as indicated by receptor autoradiography and by resistance to the hypothermic effects of the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). Homozygous mutants display a consistent pattern of responses indicative of elevated anxiety levels in open-field, elevated-zero maze, and novel-object assays. Moreover, they exhibit antidepressant-like responses in a tail-suspension assay. These results indicate that the targeted disruption of the 5-HT1A receptor gene leads to heritable perturbations in the serotonergic regulation of emotional state. 5-HT1A receptor-null mutant mice have potential as a model for investigating mechanisms through which serotonergic systems modulate affective state and mediate the actions of psychiatric drugs (Heisler, 1998).

To investigate the contribution of individual serotonin receptors to mood control, homologous recombination was used to generate mice lacking specific serotonergic receptor subtypes. Mice without 5-HT1A receptors display decreased exploratory activity and increased fear of aversive environments (open or elevated spaces). 5-HT1A knockout mice also exhibited a decreased immobility in the forced swim test, an effect commonly associated with antidepressant treatment. Although 5-HT1A receptors are involved in controlling the activity of serotonergic neurons, 5-HT1A knockout mice had normal levels of 5-HT and 5-hydroxyindoleacetic acid, possibly because of an up-regulation of 5-HT1B autoreceptors. Heterozygote 5-HT1A mutants expressed approximately one-half of wild-type receptor density and displayed intermediate phenotypes in most behavioral tests. These results demonstrate that 5-HT1A receptors are involved in the modulation of exploratory and fear-related behaviors and suggest that reductions in 5-HT1A receptor density due to genetic defects or environmental stressors might result in heightened anxiety (Ramboz, 1998).

The hippocampus is a major limbic target of the brainstem serotonergic neurons that modulate fear, anxiety, and learning through postsynaptic 5-HT1A receptors. Because chronic stress selectively down-regulates the 5-HT1A receptors in the hippocampus, it was hypothesized that mice lacking these receptors may exhibit abnormalities reminiscent of symptoms of stress-related psychiatric disorders. In particular, a hippocampal deficit in the 5-HT1A receptor could contribute to the cognitive abnormalities often seen in these disorders. To test whether a deficit in 5-HT1A receptors impairs hippocampus-related functions, hippocampal-dependent learning and memory, synaptic plasticity in the hippocampus, and limbic neuronal excitability was studied in 5-HT1A-knockout (KO) mice. 5-HT1A-KO animals showed a deficit in hippocampal-dependent learning and memory tests, such as the hidden platform (spatial) version of the Morris water maze and the delayed version of the Y maze. The performance of KO mice was not impaired in nonhippocampal memory tasks such as the visible platform (nonspatial) version of the Morris water maze, the immediate version of the Y maze, and the spontaneous-alternation test of working memory. Furthermore, paired-pulse facilitation in the dentate gyrus of the hippocampus was impaired in 5-HT1A-KO mice. Finally, 5-HT1A-KO mice, as compared with wild-type animals, displayed higher limbic excitability manifested as lower seizure threshold and higher lethality in response to kainic acid administration. These results demonstrate that 5-HT1A receptors are required for maintaining normal hippocampal functions and implicate a role for the 5-HT1A receptor in hippocampal-related symptoms, such as cognitive disturbances, in stress-related disorders (Sarnyai, 2000).

Inhibition of serotonergic raphe neurons is mediated by somatodendritic 5-HT1A autoreceptors, which may be increased in depressed patients. This study reports an association of the C(-1019)G 5-HT1A promoter polymorphism with major depression and suicide in separate cohorts. In depressed patients, the homozygous G(-1019) allele was enriched twofold versus controls, and in completed suicide cases the G(-1019) allele was enriched fourfold. The C(-1019) allele was part of a 26 bp imperfect palindrome that bound transcription factors nuclear NUDR [nuclear deformed epidermal autoregulatory factor (DEAF-1)]/suppressin and Hairy/Enhancer-of-split-5 (Drosophila) (Hes5) to repress 5-HT1A or heterologous promoters, whereas the G(-1019) allele abolished repression by NUDR, but only partially impaired Hes5-mediated repression. Recombinant NUDR bound specifically to the 26 bp palindrome, and endogenous NUDR was present in the major protein-DNA complex from raphe nuclear extracts. Stable expression of NUDR in raphe cells reduced levels of endogenous 5-HT1A protein and binding. NUDR protein was colocalized with 5-HT1A receptors in serotonergic raphe cells, hippocampal and cortical neurons, and adult brain regions including raphe nuclei, indicating a role in regulating 5-HT1A autoreceptor expression. These data indicate that NUDR is a repressor of the 5-HT1A receptor in raphe cells the function of which is abrogated by a promoter polymorphism. A novel transcriptional model is suggested in which the G(-1019) allele derepresses 5-HT1A autoreceptor expression to reduce serotonergic neurotransmission, predisposing to depression and suicide (Lemonde, 2003).

Previous studies found that serotonin transporter (SERT) knock-out mice showed increased sensitivity to minor stress and increased anxiety-like behavior but reduced locomotor activity. These mice also showed decreased density of 5-HT1A receptors in the hypothalamus, amygdala, and dorsal raphe. To evaluate the contribution of hypothalamic 5-HT1A receptors to these phenotypes of SERT knock-out mice, two studies were conducted. Recombinant adenoviruses containing 5-HT1A sense and antisense sequences (Ad-1AP-sense and Ad-1AP-antisense) were used to manipulate 5-HT1A receptors in the hypothalamus. The expression of the 5-HT1A genes is controlled by the 5-HT1A promoter, so that they are only expressed in 5-HT1A receptor-containing cells. Injection of Ad-1AP-sense into the hypothalamus of SERT knock-out mice restored 5-HT1A receptors in the medial hypothalamus; this effect was accompanied by elimination of the exaggerated adrenocorticotropin responses to a saline injection (minor stress) and reduced locomotor activity but not by a change in increased exploratory anxiety-like behavior. To further confirm the observation in SERT-/- mice, Ad-1AP-antisense was injected into the hypothalamus of normal mice. The density and the function of 5-HT1A receptors in the medial hypothalamus were significantly reduced in Ad-1AP-antisense-treated mice. Compared with a control group, Ad-1A-antisense-treated mice showed a significant reduction in locomotor activity, but again no changes in exploratory anxiety-like behaviors, tested by elevated plus-maze and open-field tests. Thus, the present results demonstrate that medial hypothalamic 5-HT1A receptors regulate stress responses and locomotor activity but may not regulate exploratory anxiety-like behaviors (Li, 2004).

Mice lacking the serotonin 1A receptor (5-HT1AR) show increased levels of anxiety-related behavior across multiple tests and background strains. Tissue-specific rescue experiments, lesion studies, and neurophysiological findings all point toward the hippocampus as a potential mediator of the phenotype. Serotonin, acting through 5-HT1ARs, can suppress hippocampal theta-frequency oscillations, suggesting that theta oscillations might be increased in the knock-outs. To test this hypothesis, local field potential recordings were obtained from the hippocampus of awake, behaving knock-outs and wild-type littermates. The magnitude of theta oscillations was increased in the knock-outs, specifically in the anxiety-provoking elevated plus maze and not in a familiar environment or during rapid eye movement sleep. Theta power correlated with the fraction of time spent in the open arms, an anxiety-related behavioral variable. These results suggest a possible role for the hippocampus, and theta oscillations in particular, in the expression of anxiety in 5-HT1AR-deficient mice (Gordon, 2005).

Serotonin receptors, circadian rhythms, and sleep

The involvement of 5-HT1B receptors in the regulation of vigilance states was assessed by investigating the spontaneous sleep-waking cycles and the effects of 5-HT receptor ligands on sleep in knock-out (5-HT1B-/-) mice that do not express this receptor type. Both 5-HT1B-/- and wild-type 129/Sv mice exhibited a clear-cut diurnal sleep-wakefulness rhythm, but knock-out animals were characterized by higher amounts of paradoxical sleep and lower amounts of slow-wave sleep during the light phase and by a lack of paradoxical sleep rebound after deprivation. In wild-type mice, the 5-HT1B agonists CP 94253 and RU 24969 induced a dose-dependent reduction of paradoxical sleep during the 2-6 hr after injection, whereas the 5-HT1B/1D antagonist GR 127935 enhanced paradoxical sleep. In addition, pretreatment with GR 127935, but not with the 5-HT1A antagonist WAY 100635, prevented the effects of both 5-HT1B agonists. In contrast, none of the 5-HT1B receptor ligands, at the same doses as those used in wild-type mice, had any effect on sleep in 5-HT1B-/- mutants. Finally, the 5-HT1A agonist 8-OH-DPAT induced in both strains a reduction in the amount of paradoxical sleep. Altogether, these data indicate that 5-HT1B receptors participate in the regulation of paradoxical sleep in the mouse (Boutrel, 1999).

Mammalian circadian rhythms are synchronized daily to light-dark cycles in the environment. The suprachiasmatic nucleus (SCN) is the proposed site of the major circadian pacemaker. Daily entrainment is believed to be influenced by inputs to the SCN, one of these being the dense serotonergic (5-HT) projection from the raphe nuclei. WAY-100635 is a potent and selective 5-HT1A receptor antagonist. In this study, the effects of WAY-100635 on phase-shifts of the hamster circadian pacemaker to light were investigated. Phase-delays after a light pulse administered during the early subjective night (15 min at CT14) were observed to be significantly greater following pre-treatment with WAY-100635 compared to light pulse alone. However, pre-treatment with WAY-100635 had no effect on the magnitude of phase-shifts to light at CT18, late in the subjective night. Serotonin may influence the responsiveness of the circadian pacemaker to photic stimuli. Specifically, WAY-100635 administered at CT14 can augment phase-shifts to light (Smart, 2001).

Selective serotonin reuptake inhibitors (SSRIs) are extensively used for the treatment of depression. Aside from their antidepressant properties, they provoke a deficit in paradoxical sleep (PS) that is most probably mediated by the transporter blockade-induced increase in serotonin concentration in the extracellular space. Such an effect can be accounted for by the action of serotonin at various types of serotonergic receptors involved in PS regulation, among which the 5-HT1A and 5-HT1B types are the best candidates. According to this hypothesis, the effects were examined of citalopram, the most selective SSRI available to date, on sleep in the mouse after inactivation of 5-HT1A or 5-HT1B receptors, either by homologous recombination of their encoding genes, or pharmacological blockade with selective antagonists. For this purpose, sleep parameters of knockout mice that do not express these receptors and their wild-type counterparts were monitored during 8 h after injection of citalopram alone or in association with 5-HT1A or 5-HT5-HT1B receptor antagonists. Citalopram induced mainly a dose-dependent inhibition of PS during 2-6 h after injection, which was observed in wild-type and 5-HT5-HT1B-/- mice, but not in 5-HT1A-/- mutants. This PS inhibition was fully antagonized by pretreatment with the 5-HT1A antagonist WAY 100635, but only partially with the 5-HT5-HT1B antagonist GR 127935. These data indicate that the action of the SSRI citalopram on sleep in the mouse is essentially mediated by 5-HT1A receptors. Such a mechanism of action provides further support to the clinical strategy of antidepressant augmentation by 5-HT1A antagonists, because the latter would also counteract the direct sleep-inhibitory side-effects of SSRIs (Monaca, 2003).

In serotonin transporter knock-out (5-HTT-/-) mice, extracellular serotonin (5-HT) levels are markedly elevated in the brain, and rapid eye movement sleep (REMS) is enhanced compared with wild-type mice. It is hypothesized that such sleep impairment at adulthood results from excessive serotonergic tone during early life. Thus, tests were performed to see if neonatal treatment with drugs capable of limiting the impact of 5-HT on the brain could normalize sleep patterns in 5-HTT-/- mutants. It was found that treatments initiated at postnatal day 5 and continued for 2 weeks with the 5-HT synthesis inhibitor para-chlorophenylalanine, or for 4 weeks with the 5-HT1A receptor (5-HT1AR) antagonist N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), induced total or partial recovery of REMS, respectively, in 5-HTT-/- mutants. Early life treatment with WAY 100635 also reversed the depression-like behavior otherwise observed in these mutants. Possible adaptive changes in 5-HT1AR after neonatal treatment with WAY 100635 were investigated by measuring 5-HT1A binding sites and 5-HT1A mRNA in various REMS- and/or depression-related brain areas, as well as 5-HT1AR-mediated hypothermia and inhibition of neuronal firing in the dorsal raphe nucleus. None of these characteristics were modified in parallel with REMS recovery, suggesting that 5-HT1ARs involved in wild-type phenotype rescue in 5-HTT-/- mutants are located in other brain areas or in 5-HT1AR-unrelated circuits where they could be transiently expressed during development. The reversal of sleep alterations and depression-like behavior after early life blockade of 5-HT1AR in 5-HTT-/- mutants might open new perspectives regarding preventive care of sleep and mood disorders resulting from serotonin transporter impairments during development (Alexandre, 2006).

Serotonin receptors regulate Glutamate receptor channels

The regulation of AMPA receptor channels by serotonin signaling in pyramidal neurons of prefrontal cortex (PFC) was studied. Application of serotonin reduced the amplitude of AMPA-evoked currents, an effect mimicked by 5-HT1A receptor agonists and blocked by 5-HT1A antagonists, indicating the mediation by 5-HT1A receptors. The serotonergic modulation of AMPA receptor currents was blocked by protein kinase A (PKA) activators and occluded by PKA inhibitors. Inhibiting the catalytic activity of protein phosphatase 1 (PP1) also eliminated the effect of serotonin on AMPA currents. Furthermore, the serotonergic modulation of AMPA currents was occluded by application of the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitors and blocked by intracellular injection of calmodulin or recombinant CaMKII. Application of serotonin or 5-HT1A agonists to PFC slices reduced CaMKII activity and the phosphorylation of AMPA receptor subunit GluR1 at the CaMKII site in a PP1-dependent manner. It is concluded that serotonin, by activating 5-HT1A receptors, suppress glutamatergic signaling through the inhibition of CaMKII, which is achieved by the inhibition of PKA and ensuing activation of PP1. This modulation demonstrates the critical role of CaMKII in serotonergic regulation of PFC neuronal activity, which may explain the neuropsychiatric behavioral phenotypes seen in CaMKII knockout mice (Cai, 2002).

The serotonin system and NMDA receptors (NMDARs) in prefrontal cortex (PFC) are both critically involved in the regulation of cognition and emotion under normal and pathological conditions; however, the interactions between them are essentially unknown. Serotonin, by activating 5-HT1A receptors, inhibits NMDA receptor-mediated ionic and synaptic currents in PFC pyramidal neurons, and the NR2B subunit-containing NMDA receptor is the primary target of 5-HT1A receptors. This effect of 5-HT1A receptors is blocked by agents that interfere with microtubule assembly, as well as by cellular knock-down of the kinesin motor protein KIF17 (kinesin superfamily member 17), which transports NR2B-containing vesicles along microtubule in neuronal dendrites. Inhibition of either CaMKII (calcium/calmodulin-dependent kinase II) or MEK/ERK (mitogen-activated protein kinase kinase/extracellular signal-regulated kinase) abolished the 5-HT1A modulation of NMDAR currents. Biochemical evidence also indicates that 5-HT1A activation reduced microtubule stability, which was abolished by CaMKII or MEK inhibitors. Moreover, immunocytochemical studies show that 5-HT1A activation decreased the number of surface NR2B subunits on dendrites, which was prevented by the microtubule stabilizer. Together, these results suggest that serotonin suppresses NMDAR function through a mechanism dependent on microtubule/kinesin-based dendritic transport of NMDA receptors that is regulated by CaMKII and ERK signaling pathways. The 5-HT1A-NMDAR interaction provides a potential mechanism underlying the role of serotonin in controlling emotional and cognitive processes subserved by PFC (Yuen, 2005).

Serotonin mediated regulation of GABAA

Benzodiazepines (BZs) acting as modulators of GABAA receptors (GABAARs) are an important group of drugs for the treatment of anxiety disorders. However, a large inter-individual variation in BZ sensitivity occurs in the human population with some anxiety disorder patients exhibiting diminished sensitivity to BZ and reduced density of GABAARs. The mechanism underlying BZ treatment resistance is not known, and it is not possible to predict whether an anxiety patient will respond to BZ. 5-hydroxytryptamine1A receptor (5-HT1AR) null mice (R-/-) on the Swiss-Webster (SW) background reproduce several features of BZ-resistant anxiety; they exhibit anxiety-related behaviors, do not respond to BZ, have reduced BZ binding, and have decreased expression of the major GABAAR subunits alpha1 and alpha2. R-/- mice on the C57Bl6 (B6) background also have anxiety phenotype, but they respond to BZ and have normal GABAAR subunit expression. This indicates that the 5-HT1AR-mediated regulation of GABAAR alpha subunit expression is subject to genetic modification. Hybrid SW/B6-R-/- mice also exhibit BZ-resistant anxiety, suggesting that SW mice carry a genetic modifier, which mediates the effect of the 5-HT1AR on the expression of GABAARalpha subunits. In addition, this genetic interaction in SW mice operates early in postnatal life to influence the expression of GABAAR alpha subunits at the transcriptional level. These data indicate that BZ-resistant anxiety results from a developmental arrest of GABAAR expression in SW-R-/- mice, and a similar mechanism may be responsible for the BZ insensitivity of some anxiety patients (Bailey, 2004).

Effects of antipsychotic drugs acting through serotonin receptors

The prefrontal cortex plays a key role in the control of higher brain functions and is involved in the pathophysiology and treatment of schizophrenia. Approximately 60% of the neurons in rat and mouse prefrontal cortex express 5-HT1A and/or 5-HT2A receptor mRNAs, which are highly co-localized (approximately 80%). The electrical stimulation of the dorsal and median raphe nuclei elicited 5-HT1A-mediated inhibitions and 5-HT2A-mediated excitations in identified pyramidal neurons recorded extracellularly in rat medial prefrontal cortex (mPFC). Opposite responses in the same pyramidal neuron could be evoked by stimulating the raphe nuclei at different coordinates, suggesting a precise connectivity between 5-HT neuronal subgroups and 5-HT1A and 5-HT2A receptors in pyramidal neurons. Microdialysis experiments showed that the increase in local 5-HT release evoked by the activation of 5-HT2A receptors in mPFC by DOI (5-HT2A/2C receptor agonist) was reversed by co-perfusion of 5-HT1A agonists. This inhibitory effect was antagonized by WAY-100635 and the prior inactivation of 5-HT1A receptors in rats and was absent in mice lacking 5-HT1A receptors. These observations help to clarify the interactions between the mPFC and the raphe nuclei, two key areas in psychiatric illnesses and improve understanding of the action of atypical antipsychotics, acting through these 5-HT receptors (Amargos-Bosch, 2004).

Both orexin and serotonin (5-HT) have important roles in the regulation of sleep-wakefulness, as well as in feeding behavior. The effects of 5-HT on orexin/hypocretin neurons was examined, using hypothalamic slices prepared from orexin/enhanced green fluorescent protein (EGFP) transgenic mice in which EGFP is expressed exclusively in orexin neurons. Patch-clamp recording from EGFP-expressing cells showed that 5-HT hyperpolarized all orexin neurons in a concentration-dependent manner. The response was inhibited by the 5-HT1A receptor antagonist WAY100635. A 5-HT1A receptor agonist, 8-hydroxy-2-(dl-N-propyl-amino)tetralin, also evoked hyperpolarization on orexin neurons with potency comparable with 5-HT. A low concentration of Ba2+ (30 microM) inhibited 5-HT-induced hyperpolarization. Single-channel recording revealed that the conductance of 5-HT-induced channel activity was 33.8 pS, which is in good agreement with that of the G-protein-coupled inward rectifier potassium channel (GIRK). Moreover, 5-HT1A receptor-like immunoreactivity was observed on orexin neurons, and 5-HT transporter immunoreactive nerve endings are in close apposition to orexin neurons. Intracerebroventricular injection of the 5-HT1A receptor-selective antagonist WAY100635 increased locomotor activity during the latter half of dark phase in wild-type mice but not in orexin/ataxin-3 mice in which orexin neurons are specifically ablated, suggesting that activation of orexin neurons is necessary for the WAY100635-induced increase in locomotor activity. These results indicate that 5-HT hyperpolarizes orexin neurons through the 5-HT1A receptor and subsequent activation of the GIRK and that this inhibitory serotonergic input to the orexin neurons is likely to be important for the physiological regulation of this neuropeptide system (Muraki, 2004).


REFERENCES

Search PubMed for articles about Drosophila Serotonin receptor 1A and Serotonin receptor 1B

Search PubMed for articles about Drosophila 5-HT1A and 5-HT1B

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date revised: 15 February 2011

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