InteractiveFly: GeneBrief

Dopamine 1-like receptor 1 : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - Dopamine 1-like receptor 1

Synonyms - dDA1, Dopamine receptor, DopR, Dumb

Cytological map position-88A10-88A12

Function - receptor

Keywords - behavior, mediation of aversive and appetitive Pavlovian conditioning

Symbol - Dop1R1

FlyBase ID: FBgn0011582

Genetic map position - 3R: 10,003,005..10,040,409 [-]

Classification - G-protein coupled receptor

Cellular location - transmembrane

NCBI link: EntrezGene
Dop1R1 orthologs: Biolitmine

Recent literature
Pitmon, E., Stephens, G., Parkhurst, S. J., Wolf, F. W., Kehne, G., Taylor, M. and Lebestky, T. (2016). The D1 Family Dopamine Receptor, DopR, potentiates hindleg grooming behavior in Drosophila. Genes Brain Behav [Epub ahead of print]. PubMed ID: 26749475
Drosophila groom away debris and pathogens from the body using their legs in a stereotyped sequence of innate motor behaviors. This study investigated one aspect of the grooming repertoire by characterizing the D1 family dopamine receptor DopR. Removal of DopR results in decreased hindleg grooming, as substantiated by quantitation of dye remaining on mutant and RNAi animals versus controls and direct scoring of behavioral events. These data are also supported by pharmacological results that D1 receptor agonists fail to potentiate grooming behaviors in headless DopR flies. DopR protein is broadly expressed in the neuropil of the thoracic ganglion and overlaps with TH-positive dopaminergic neurons. Broad neuronal expression of Dopamine Receptor in mutant animals restored normal grooming behaviors. These data provide evidence for the role of DopR in potentiating hindleg grooming behaviors in the thoracic ganglion of adult Drosophila. This is a remarkable juxtaposition to the considerable role of D1 family dopamine receptors in rodent grooming, and future investigations of evolutionary relationships of circuitry may be warranted.

Zhang, Y., Guo, J., Guo, A. and Li, Y. (2016). Nicotine-induced acute hyperactivity is mediated by dopaminergic system in a sexually dimorphic manner. Neuroscience [Epub ahead of print]. PubMed ID: 27365175
Short-term exposure to nicotine induces positive effects in mice, monkeys and humans, including mild euphoria, hyperactivity, and enhanced cognition.Using a video recording system, this study found that acute nicotine administration induces locomotor hyperactivity in Drosophila. Suppressing dopaminergic neurons or down-regulating Dopamine 1-like receptor (DopR) abolishes this acute nicotine response, but surprisingly, does so only in male flies. Using a GFP reconstitution across synaptic partners (GRASP) approach, dopaminergic neurons were shown to possess potential synaptic connections with acetylcholinergic neurons in wide regions of the brain. Furthermore, dopaminergic neurons are widely activated upon nicotine perfusion in both sexes, while the response curve differs significantly between the sexes. Moreover, knockdown of the β1 nicotine acetylcholine receptor (nAChR) in dopaminergic neurons abolishes the acute nicotine response only in male flies, while panneural knock-down occurs in both sexes. Taken together, these results reveal that in fruit flies, dopaminergic neurons mediate nicotine-induced acute locomotor hyperactivity in a sexually dimorphic manner, and Drosophila β1 nAChR subunit plays a crucial role in this nicotine response.
Jiang, Y., Pitmon, E., Berry, J., Wolf, F. W., McKenzie, Z. and Lebestky, T. J. (2016). A genetic screen to assess Dopamine receptor (DopR1) dependent sleep regulation in Drosophila. G3 (Bethesda) [Epub ahead of print]. PubMed ID: 27760793
Sleep is an essential behavioral state of rest that is regulated by homeostatic drives to ensure a balance of sleep and activity, as well as independent arousal mechanisms in the central brain. Dopamine has been identified as a critical regulator of both sleep behavior and arousal. This study presents results of a genetic screen that selectively restored the Dopamine Receptor (DopR/DopR1/dumb) to specific neuroanatomical regions of the adult Drosophila brain to assess requirements for DopR in sleep behavior. Subsets of the mushroom body were identified that utilize DopR in daytime sleep regulation. These data are supported by multiple examples of spatially restricted genetic rescue data in discrete circuits of the mushroom body, as well as immunohistochemistry that corroborates the localization of DopR protein within mushroom body circuits. Independent loss of function data using an inducible RNAi construct in the same specific circuits also supports a requirement for DopR in daytime sleep. Additional circuit activation of discrete DopR+ mushroom body neurons also suggests roles for these subpopulations in sleep behavior. These conclusions support a new separable function for DopR in daytime sleep regulation within the mushroom body. This daytime regulation is independent of the known role of DopR in nighttime sleep, which is regulated within the Fan Shaped Body. This study provides new neuroanatomical loci for exploration of dopaminergic sleep functions in Drosophila, and expands understanding of sleep regulation during the day versus night.
Lim, J., Fernandez, A. I., Hinojos, S. J., Aranda, G. P., James, J., Seong, C. S. and Han, K. A. (2017). The mushroom body D1 dopamine receptor controls innate courtship drive. Genes Brain Behav [Epub ahead of print]. PubMed ID: 28902472
Mating is critical for species survival and is profoundly regulated by neuromodulators and neurohormones to accommodate internal states and external factors. To identify the underlying neuromodulatory mechanisms, this study investigated the roles of dopamine receptors in various aspects of courtship behavior in Drosophila. The D1 dopamine receptor dDA1 regulates courtship drive in naive males. The wild-type naive males actively courted females regardless their appearance or mating status. On the contrary, the dDA1 mutant (dumb) males exhibited substantially reduced courtship toward less appealing females including decapitated, leg-less and mated females. The dumb male's reduced courtship activity was due to delay in courtship initiation and prolonged intervals between courtship bouts. The dampened courtship drive of dumb males was rescued by reinstated dDA1 expression in the mushroom body α/&beta& and γ neurons but not α/β or γ neurons alone, which is distinct from the previously characterized dDA1 functions in experience-dependent courtship or other learning and memory processes. It was also found that the dopamine receptors dDA1, DAMB and dD2R are dispensable for associative memory formation and short-term memory of conditioned courtship, thus courtship motivation and associative courtship learning and memory are regulated by distinct neuromodulatory mechanisms. Taken together, this study narrows the gap in the knowledge of the mechanism that dopamine regulates male courtship behavior.
Lau, M. T., Lin, Y. Q., Kisling, S., Cotterell, J., Wilson, Y. A., Wang, Q. P., Khuong, T. M., Bakhshi, N., Cole, T. A., Oyston, L. J., Cole, A. R. and Neely, G. G. (2017). A simple high throughput assay to evaluate water consumption in the fruit fly. Sci Rep 7(1): 16786. PubMed ID: 29196744
Water intake is essential for survival and thus under strong regulation. This study describes a simple high throughput system to monitor water intake over time in Drosophila. The design of the assay involves dehydrating fly food and then adding water back separately so flies either eat or drink. Water consumption is then evaluated by weighing the water vessel and comparing this back to an evaporation control. This system is high throughput, does not require animals to be artificially dehydrated, and is simple both in design and implementation. Initial characterisation of homeostatic water consumption shows high reproducibility between biological replicates in a variety of experimental conditions. Water consumption was dependent on ambient temperature and humidity and was equal between sexes when corrected for mass. By combining this system with the Drosophila genetics tools, it was possible to confirm a role for ppk28 and DopR1 in promoting water consumption, and through functional investigation of RNAseq data from dehydrated animals, it was found that DopR1 expression in the mushroom body was sufficient to drive consumption and enhance water taste sensitivity. Together, this study provides a simple high throughput water consumption assay that can be used to dissect the cellular and molecular machinery regulating water homeostasis in Drosophila.
Kottler, B., Faville, R., Bridi, J. C. and Hirth, F. (2019). Inverse control of turning behavior by Dopamine D1 receptor signaling in columnar and ring neurons of the central complex in Drosophila. Curr Biol. PubMed ID: 30713106
Action selection is a prerequisite for decision-making and a fundamental aspect to any goal-directed locomotion; it requires integration of sensory signals and internal states to translate them into action sequences. This paper introduces a novel behavioral analysis to study neural circuits and mechanisms underlying action selection and decision-making in freely moving Drosophila. Preferred patterns of motor activity and turning behavior were discovered. These patterns are impaired in FoxP mutant flies, which present an altered temporal organization of motor actions and turning behavior, reminiscent of indecisiveness. Then, focusing on central complex (CX) circuits known to integrate different sensory modalities and controlling premotor regions, action sequences and turning behavior were shown to be regulated by dopamine D1-like receptor (Dop1R1) signaling. Dop1R1 inputs onto CX columnar ellipsoid body-protocerebral bridge gall (E-PG) neuron and ellipsoid body (EB) R2/R4m ring neuron circuits both negatively gate motor activity but inversely control turning behavior. Although flies deficient of D1 receptor signaling present normal turning behavior despite decreased activity, restoring Dop1R1 level in R2/R4m-specific circuitry affects the temporal organization of motor actions and turning. EB R2/R4m neurons are in contact with E-PG neurons that are thought to encode body orientation and heading direction of the fly. These findings suggest that Dop1R1 signaling in E-PG and EB R2/4 m circuits are compared against each other, thereby modulating patterns of activity and turning behavior for goal-directed locomotion.
Silva, B., Hidalgo, S. and Campusano, J. M. (2020). Dop1R1, a type 1 dopaminergic receptor expressed in Mushroom Bodies, modulates Drosophila larval locomotion. PLoS One 15(2): e0229671. PubMed ID: 32101569
As in vertebrates, dopaminergic neural systems are key regulators of motor programs in insects, including the fly Drosophila melanogaster. Dopaminergic systems innervate the Mushroom Bodies (MB), an important association area in the insect brain primarily associated to olfactory learning and memory, but that has been also implicated with the execution of motor programs. The main objectives of this work is to assess the idea that dopaminergic systems contribute to the execution of motor programs in Drosophila larvae, and then, to evaluate the contribution of specific dopaminergic receptors expressed in MB to these programs. The results show that animals bearing a mutation in the dopamine transporter show reduced locomotion, while mutants for the dopaminergic biosynthetic enzymes or the dopamine receptor Dop1R1 exhibit increased locomotion. Pan-neuronal expression of an RNAi for the Dop1R1 confirmed these results. Further studies show that animals expressing the RNAi for Dop1R1 in the entire MB neuronal population or only in the MB gamma-lobe forming neurons, exhibit an increased motor output, as well. Interestingly, these results also suggest that other dopaminergic receptors do not contribute to larval motor behavior. Thus, the data support the proposition that CNS dopamine systems innervating MB neurons modulate larval locomotion and that Dop1R1 mediates this effect.
Sun, R., Delly, J., Sereno, E., Wong, S., Chen, X., Wang, Y., Huang, Y. and Greenspan, R. J. (2020). Anti-instinctive Learning Behavior Revealed by Locomotion-Triggered Mild Heat Stress in Drosophila. Front Behav Neurosci 14: 41. PubMed ID: 32372923
Anti-instinctive learning, an ability to modify an animal's innate behaviors in ways that go against one's innate tendency, can confer great evolutionary advantages to animals and enable them to better adapt to the changing environment. Yet, understanding of anti-instinctive learning and its underlying mechanisms is still limited. This work describes a new anti-instinctive learning behavior of fruit flies. This learning paradigm requires the fruit fly to respond to a recurring, aversive, mild heat stress by modifying its innate locomotion behavior. Experiencing movement-triggered mild heat stress repeatedly significantly reduced walking activity in wild type fruit flies, indicating that fruit flies are capable of anti-instinctive learning. This study also reports that such learning ability is reduced in dopamine 1-like receptor 1 (Dop1R1) null mutant and dopamine 2-like receptor (Dop2R) null mutant flies, suggesting that these two dopamine receptors are involved in mediating anti-instinctive learning in flies.
Fernandez-Chiappe, F., Hermann-Luibl, C., Peteranderl, A., Reinhard, N., Senthilan, P. R., Hieke, M., Selcho, M., Yoshii, T., Shafer, O. T., Muraro, N. I. and Helfrich-Forster, C. (2020). Dopamine signaling in wake promoting clock neurons is not required for the normal regulation of sleep in Drosophila. J Neurosci. PubMed ID: 33172977
Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake- and sleep-promoting cell types. The large and small ventrolateral neurons (l-LN(v)s and s-LN(v)s) have been identified as wake-promoting neurons within the clock neuron network. The l-LN(v)s are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LN(v)s and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. This study tested this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. Dopamine application indeed increases cAMP levels and depolarizes the l-LN(v)s, but surprisingly, it does not result in increased firing rates. Down-regulation of the excitatory dopamine receptor, Dop1R1 in the l- and s-LN(v)s, but not of Dop1R2, abolished the depolarization of l-LN(v)s in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LN(v)s. Down-regulation of Dop1R1 or Dop1R2 receptors in the l- and s-LN(v)s does not affect sleep in males. Unexpectedly, a moderate decrease of daytime sleep was found with down-regulation of Dop1R1 and of nighttime sleep with down-regulation of Dop1R2. Since the l-LN(v)s do not utilize Dop1R2 receptors and the s-LN(v)s also respond to dopamine, it is concluded that the s-LN(v)s are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LN(v)s is not required for daytime arousal, but likely promotes nighttime sleep via the s-LN(v)s.
Zhang, R., Du, J., Zhao, X., Wei, L. and Zhao, Z. (2020). Regulation of circadian behavioural output via clock-responsive miR-276b. Insect Mol Biol. PubMed ID: 33131172
Growing evidence indicates that microRNAs play numerous important roles. However, the roles of some microRNAs involved in regulation of circadian rhythm and sleep are still not well understood. This study shows that the miR-276b is essential for maintaining both sleep and circadian rhythm by targeting tim, NPFR and DopR1 genes, with miR-276b deleted mutant flies sleeping more, and vice versa in miR-276b overexpressing flies. Through analysing its promoter, mir-276b was found to be responsive to CLOCK and regulates circadian rhythm through the negative feedback loop of the CLK/CYC-TIM/PER. Furthermore, miR-276b is broadly expressed in the clock neurons and the central complexes such as the mushroom body and the fan-shape body of Drosophila brain, in which up-regulation of miR-276b in tim, npfr1 and DopR1 expressing tissues significantly causes sleep decreases. This study clarifies that the mir-276b is very important for participating in regulation of circadian rhythm and sleep.
Kanno, M., Hiramatsu, S., Kondo, S., Tanimoto, H. and Ichinose, T. (2021). Voluntary intake of psychoactive substances is regulated by the dopamine receptor Dop1R1 in Drosophila. Sci Rep 11(1): 3432. PubMed ID: 33564023
Dysregulated motivation to consume psychoactive substances leads to addictive behaviors that often result in serious health consequences. Understanding the neuronal mechanisms that drive drug consumption is crucial for developing new therapeutic strategies. The fruit fly Drosophila melanogaster offers a unique opportunity to approach this problem with a battery of sophisticated neurogenetic tools available, but how they consume these drugs remains largely unknown. This study examined drug self-administration behavior of Drosophila and the underlying neuronal mechanisms. The preference of flies for five different psychoactive substances was measured using a two-choice feeding assay and its long-term changes were monitored. Flies were found to show acute preference for ethanol and methamphetamine, but not for cocaine, caffeine or morphine. Repeated intake of ethanol, but not methamphetamine, increased over time. Preference for methamphetamine and the long-term escalation of ethanol preference required the dopamine receptor Dop1R1 in the mushroom body. The protein level of Dop1R1 increased after repeated intake of ethanol, but not methamphetamine, which correlates with the acquired preference. Genetic overexpression of Dop1R1 enhanced ethanol preference. These results reveal a striking diversity of response to individual drugs in the fly and the role of dopamine signaling and its plastic changes in controlling voluntary intake of drugs.
El Kholy, S., Wang, K., El-Seedi, H. R. and Al Naggar, Y. (2021). Dopamine Modulates Drosophila Gut Physiology, Providing New Insights for Future Gastrointestinal Pharmacotherapy. Biology (Basel) 10(10). PubMed ID: 34681083.
Dopamine has a variety of physiological roles in the gastrointestinal tract (GI) through binding to Drosophila dopamine D1-like receptors (DARs) and/or adrenergic receptors and has been confirmed as one of the enteric neurotransmitters. To gain new insights into what could be a potential future promise for GI pharmacology, Drosophila was used as a model organism to investigate the effects of dopamine on intestinal physiology and gut motility. GAL4/UAS system was utilized to knock down specific dopamine receptors using specialized GAL4 driver lines targeting neurons or enterocytes cells to identify which dopamine receptor controls stomach contractions. DARs (Dop1R1 and Dop1R2) were shown by immunohistochemistry to be strongly expressed in all smooth muscles in both larval and adult flies, which could explain the inhibitory effect of dopamine on GI motility. Adult males' gut peristalsis was significantly inhibited by knocking down dopamine receptors Dop1R1, Dop1R2, and Dop2R, but female flies' gut peristalsis was significantly repressed by knocking down only Dop1R1 and Dop1R2. These findings also showed that dopamine drives PLC-β translocation from the cytoplasm to the plasma membrane in enterocytes for the first time. Overall, these data revealed the role of dopamine in modulating Drosophila gut physiology, offering us new insights for the future gastrointestinal pharmacotherapy of neurodegenerative diseases associated with dopamine deficiency.
Grover, D., Chen, J. Y., Xie, J., Li, J., Changeux, J. P. and Greenspan, R. J. (2022). Differential mechanisms underlie trace and delay conditioning in Drosophila.. Nature 603(7900): 302-308. PubMed ID: 35173333
Two forms of associative learning-delay conditioning and trace conditioning-have been widely investigated in humans and higher-order mammals. In delay conditioning, an unconditioned stimulus (for example, an electric shock) is introduced in the final moments of a conditioned stimulus (for example, a tone), with both ending at the same time. In trace conditioning, a 'trace' interval separates the conditioned stimulus and the unconditioned stimulus. Trace conditioning therefore relies on maintaining a neural representation of the conditioned stimulus after its termination (hence making distraction possible), to learn the conditioned stimulus-unconditioned stimulus contingency; this makes it more cognitively demanding than delay conditioning. By combining virtual-reality behaviour with neurogenetic manipulations and in vivo two-photon brain imaging, this study shows that visual trace conditioning and delay conditioning in Drosophila mobilize R2 and R4m ring neurons in the ellipsoid body. In trace conditioning, calcium transients during the trace interval show increased oscillations and slower declines over repeated training, and both of these effects are sensitive to distractions. Dopaminergic activity accompanies signal persistence in ring neurons, and this is decreased by distractions solely during trace conditioning. Finally, dopamine D1-like and D2-like receptor signalling in ring neurons have different roles in delay and trace conditioning; dopamine D1-like receptor 1 mediates both forms of conditioning, whereas the dopamine D2-like receptor is involved exclusively in sustaining ring neuron activity during the trace interval of trace conditioning.
Pan, Y., Li, W., Deng, Z., Sun, Y., Ma, X., Liang, R., Guo, X., Sun, Y., Li, W., Jiao, R. and Xue, L. (2022). Myc suppresses male-male courtship in Drosophila. Embo j 41(7): e109905. PubMed ID: 35167135
Despite strong natural selection on species, same-sex sexual attraction is widespread across animals, yet the underlying mechanisms remain elusive. This study reports that the proto-oncogene Myc is required in dopaminergic neurons to inhibit Drosophila male-male courtship. Loss of Myc, either by mutation or neuro-specific knockdown, induced males' courtship propensity toward other males. Genetic screen identified DOPA decarboxylase (Ddc) as a downstream target of Myc. While loss of Ddc abrogated Myc depletion-induced male-male courtship, Ddc overexpression sufficed to trigger such behavior. Furthermore, Myc-depleted males exhibited elevated dopamine level in a Ddc-dependent manner, and their male-male courtship was blocked by depleting the dopamine receptor DopR1. Moreover, Myc directly inhibits Ddc transcription by binding to a target site in the Ddc promoter, and deletion of this site by genome editing was sufficient to trigger male-male courtship. Finally, drug-mediated Myc depletion in adult neurons by GeneSwitch technique sufficed to elicit male-male courtship. Thus, this study uncovered a novel function of Myc in preventing Drosophila male-male courtship, and supports the crucial roles of genetic factors in inter-male sexual behavior.
Naganos, S., Ueno, K., Horiuchi, J. and Saitoe, M. (2022). Dopamine activity in projection neurons regulates short-lasting olfactory approach memory in Drosophila. Eur J Neurosci 56(5): 4558-4571. PubMed ID: 35815601
Survival in many animals requires the ability to associate certain cues with danger and others with safety. In a Drosophila melanogaster aversive olfactory conditioning paradigm, flies are exposed to two odours, one presented coincidentally with electrical shocks, and a second presented 45 s after shock cessation. When flies are later given a choice between these two odours, they avoid the shock-paired odour and prefer the unpaired odour. While many studies have examined how flies learn to avoid the shock-paired odour through formation of odour-fear associations, this study demonstrates that conditioning also causes flies to actively approach the second odour. In contrast to fear memories, which are longer lasting and requires activity of D1-like dopamine receptors only in the mushroom bodies, approach memory is short-lasting and requires activity of D1-like dopamine receptors in projection neurons originating from the antennal lobes, primary olfactory centers. Further, while recall of fear memories requires activity of the mushroom bodies, recall of approach memories does not. These data suggest that olfactory approach memory is formed using different mechanisms in different brain locations compared to aversive and appetitive olfactory memories.

Drosophila has robust behavioral plasticity to avoid or prefer the odor that predicts punishment or food reward, respectively. Both types of plasticity are mediated by the mushroom body (MB) neurons in the brain, in which various signaling molecules play crucial roles. However, important yet unresolved molecules are the receptors that initiate aversive or appetitive learning cascades in the MB. D1 dopamine receptor dDA1 (FlyBase name: Dopamine receptor) has been shown to be highly enriched in the MB neuropil. This study demonstrates that dDA1 is a key receptor that mediates both aversive and appetitive learning in pavlovian olfactory conditioning. Two mutants, dumb1 and dumb2, have abnormal dDA1 expression. When trained with the same conditioned stimuli, both dumb alleles showed negligible learning in electric shock-mediated conditioning while they exhibited moderately impaired learning in sugar-mediated conditioning. These phenotypes are not attributable to anomalous sensory modalities of dumb mutants because their olfactory acuity, shock reactivity, and sugar preference are comparable to those of control lines. Remarkably, the dumb mutant's impaired performance in both paradigms is fully rescued by reinstating dDA1 expression in the same subset of MB neurons, indicating the critical roles of the MB dDA1 in aversive as well as appetitive learning. Previous studies using dopamine receptor antagonists implicate the involvement of D1/D5 receptors in various pavlovian conditioning tasks in mammals; however, these have not been supported by the studies of D1- or D5-deficient animals. The findings described in this study here unambiguously clarify the critical roles of D1 dopamine receptor in aversive and appetitive pavlovian conditioning (Kim, 2007b).

Pavlovian (classical) olfactory conditioning in Drosophila tests the animal's ability to learn and remember the odor [conditioned stimulus (CS)] associated with diverse unconditioned stimuli (US) and is instrumental in investigating the neural and cellular mechanisms underlying distinct learning and memory processes. When subjected to concurrent odor (CS+) and electric shock (aversive US) presentation, flies learn to avoid the CS+ odor in the absence of shock. Conversely, flies learn to prefer the CS+ odor after concurrent odor (CS+) and sugar (appetitive US) exposure. Thus, the same CS+ triggers either avoidance or preference behavior depending on previous experience of the flies. Several key questions arise regarding the underlying mechanisms. Specifically, are common or separate neural systems required for aversive versus appetitive learning and memory? What are the critical molecular and cellular events that distinguish reward versus punishment information (Kim, 2007b)?

Two key components are essential for both aversive and appetitive conditioning. One component is the cAMP signaling pathway. Flies defective in cAMP metabolism, such as dunce (cAMP-specific phosphodiesterase) and rutabaga [rut; calcium/calmodulin (CaM)-dependent adenylyl cyclase (AC)], or flies with altered activities of cAMP effectors Protein kinase A and dCREB2 are impaired in learning and/or memory in aversive conditioning. Likewise, appetitive conditioning requires cAMP because rut mutants display poor learning. The other component is the mushroom body (MB) brain structure. Flies with ablated MB structures or functions are completely defective in aversive learning. Moreover, synaptic output of different MB lobes is involved in memory formation or retrieval in aversive and appetitive conditioning. These indicate the MB as a central neural substrate for olfactory learning and memory. This poses a fundamental question regarding the neuromodulators and their receptors that initiate the cAMP cascade in the MB for aversive and appetitive learning and their memories (Kim, 2007b).

The neuromodulators that are crucial for olfactory conditioning and activate cAMP increases are dopamine and octopamine (see Tyramine β hydroxylase). Previous studies of Drosophila larvae and adults show that dopaminergic neuronal activities are essential for aversive, but not for appetitive, learning, whereas octopamine or octopaminergic neuronal activities are necessary only for appetitive learning (Schwaerzel, 2003; Schroll, 2006). Consistently, the activities of dopaminergic neurons projecting to the MB are mildly increased by odor stimuli and strongly by electric shock (Riemensperger, 2005). Moreover, duration of their activities is prolonged when the CS+ odor is presented, suggesting the role of dopamine neurons in US prediction. However, it is yet unknown whether dopamine directly activates the MB for aversive learning. To uncover the signal(s) activating the learning and memory cascade, three receptors have been identifed that are highly enriched in the MB and increase cAMP levels, and they are two dopamine receptors, dDA1 and DAMB, and an octopamine receptor, OAMB (Han, 1996; Han, 1998; Kim, 2003). This study shows that dDA1 is required in the MB for aversive and appetitive learning (Kim, 2007b).

Previous research on olfactory conditioning in Drosophila has primarily focused on the intracellular components, many of which are involved in learning and/or memory processes in the MB. Although those studies have revealed important insights, the receptors that initiate signaling cascades into motion in the MB are unknown. The findings presented here provide the first demonstration of a MB receptor essential for aversive learning. The role of dDA1 in this behavioral plasticity is physiological, rather than developmental. This is consistent with the observations that synaptic output of dopaminergic neurons is necessary during training (Schwaerzel, 2003), and the learning phenotype of rut mutants is rescued by the restricted expression of rut-AC, a potential dDA1 effector, in the adult MB (McGuire, 2003; Mao, 2004). Moreover, the dopaminergic processes projecting to the MB gamma lobe strongly respond to electric shock (US) and show altered activities after CS+ exposure (Riemensperger, 2005). Together, these strongly implicate dDA1 as a receptor conveying aversive US information in the MB lobes for memory formation (Kim, 2007b).

Two additional neuromodulator systems are previously implicated in aversive olfactory conditioning in Drosophila. One neuromodulator system is the glutamate NMDA receptor composed of dNR1 and dNR2 subunits. Flies with decreased dNR1 expression show diminished performance in aversive conditioning. Although dNR1 in the MB is crucial for anesthesia-resistant and midterm memories, dNR1-dependent learning occurs outside of the MB. Another putative modulator involved in olfactory learning is Amn, which has sequence homology with mammalian neuropeptide PACAP. Although amn mutants are mostly defective in midterm memory, they are mildly impaired in learning when BA is used as CS+ and learning of BA depends on synaptic output of Amn-expressing DPM neurons projecting to the MB lobes. Thus, it has been suggested that putative amn-encoded neuropeptides, by binding to their receptor(s) in the MB neuropil, may mediate memory formation; however, the predicted Amn neuropeptides or their receptors remain unidentified. Therefore, dDA1 represents the only MB receptor identified to date that is essential for aversive learning. Notably, dumb mutants, similar to MB-less flies, show negligible learning. This indicates that the MB neurons absolutely require dDA1 for aversive memory formation (Kim, 2007b).

The data presented in this study demonstrate the crucial role of dDA1 in sugar-mediated olfactory learning as well. Interestingly, dumb mutants have diminished, yet significant, performance scores, implicating an additional receptor(s) for this type of learning. Indeed, tßh mutants lacking octopamine show severe impairment in appetitive conditioning, which is rescued by feeding octopamine before, but not after, training (Schwaerzel, 2003). Thus, octopamine represents another neuromodulator crucial for appetitive learning. Because the MB is a primary neural substrate for appetitive conditioning, reward memory formation is likely mediated by dDA1 and an octopamine receptor(s) in the MB (Kim, 2007b).

The previous study of TH-GAL4/UAS-Shits flies, in which endocytosis of the dopamine neurons expressing TH-GAL4 can be temporally controlled by dominant-negative dynamin Shits, suggests that dopamine is not involved in appetitive conditioning (Schwaerzel, 2003; Kim, 2007a). This is contrary to the learning phenotype of D1 dopamine receptor mutants dumb. Nonetheless, the discrepancy may be reconciled by several reasonable possibilities: (1) TH-GAL4 used in the previous study to drive Shits may not be expressed, or expressed at low levels, in a subset of the dopamine neurons critical for appetitive learning; (2) dopamine neuronal output conveying sugar information may not be completely inhibited by Shits; (3) dopamine crucial for appetitive learning may be secreted by a dynamin-independent pathway. These possibilities may be tested by investigating pale mutants that are unable to synthesize dopamine; however, such flies die during development because of an essential role of dopamine in cuticle formation (Budnik, 1987). Future studies on conditional pale mutants should help resolve this issue (Kim, 2007b).

dDA1 expression driven by MB247-GAL4 fully rescues the learning phenotypes of dumb mutants in electric shock- as well as sugar-mediated conditioning. This indicates that appetitive and aversive memory formations are mediated by dDA1 in the same subset of the MB neurons (~30% of all MB neurons). This poses an intriguing question as to how those MB neurons distinguish punishment versus reward information delivered by dDA1 to generate avoidance versus preference behavioral output. The key to answering this question may be intracellular effectors in the MB neuropil. rut-AC is crucial in the MB247-GAL4-expressing MB neurons for both aversive and appetitive learning (Schwaerzel, 2003). Notably, rut mutants retain some learning capacities in electric shock- and sugar-mediated conditioning, whereas MB-less flies or the flies with inhibited MB synaptic output exhibit no trace of learning in both assays. This implicates additional cellular components crucial for aversive and appetitive memory formation. Because dumb mutants are rather completely impaired in aversive learning, dDA1 may activate rut-AC and other cellular components in the MB to process punishment information (Kim, 2007b).

G-protein-coupled receptors including dopamine receptors can recruit multiple effector systems through heteromeric G-proteins or through cross-interactions of diverse signaling components. Thus, for aversive learning, dDA1 activated by electric shock US input may recruit the mitogen-activated protein (MAP) kinase cascade in addition to the cAMP pathway. The activated protein kinase A and MAP kinases may act on ion channels or cell adhesion molecules such as integrin and FasII to modify MB synaptic output, leading to avoidance behavior. Consistently, the flies defective in 14-3-3 and S6KII, which are involved in the MAP kinase cascade, and α-integrin and fasII mutants are poor learners in electric shock-mediated conditioning (Kim, 2007b).

For reward-mediated learning, reward US input may impinge on at least two receptors, dDA1 and an octopamine receptor, in the MB. Their simultaneous activities may recruit multiple effectors that possibly include rut-AC, MAP kinases, protein kinase C, and CaM kinase II. The biochemical changes collectively activated by these effectors may alter MB synaptic output to generate preference behavior. Interestingly, OAMB (octopamine receptor) activates the increases in intracellular calcium as well as cAMP (Han, 1998) and is a good candidate that can turn on the aforementioned effectors for processing reward information in the MB. The punishment and reward effectors may be at work in separate areas of the same MB neuropil or in different MB neurons or neuropils, which are differentially innervated by dopaminergic axons conveying electric shock input or by dopaminergic and octopaminergic axons conveying sugar input. At present, there is limited information on intracellular components involved in appetitive learning. Future studies in this venue will help attest this model. Together, concurrent CS+ and US received during training may activate dDA1 (for punishment US) or dDA1 and an octopamine receptor (for reward US) to induce distinctive biochemical changes, leading to avoidance or preference behavior, respectively (Kim, 2007b).

Multiple lines of evidence indicate that dopamine in the amygdala, the nucleus accumbens, and the medial prefrontal cortex in mammals is crucial for acquisition, expression, and/or extinction in aversive pavlovian conditioning (for review, see Pezze, 2004). However, the receptors mediating the functions of dopamine are unclear. Studies using D1/D5 dopamine receptor antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH23390) in rats suggest the significant roles of the D1-type receptor in the amygdala and the nucleus accumbens during acquisition in fear conditioning and conditioned taste aversion (CTA), respectively (Guarraci, 1999; Fenu, 2001). However, the mice lacking D1 or D5 receptor show normal acquisition in fear conditioning (El-Ghundi, 2001; Holmes, 2001). Likewise, D1-deficient mice show normal learning of CTA to salt (CS) paired with LiCl (US), although they do not develop CTA to sucrose (Cannon, 2005). The discrepant findings of the pharmacological and genetic studies may be attributable to either other receptor types affected by SCH23390 or compensatory adaptations in D1 or D5 knock-out mice. The studies reported in this study support the latter and clarify the indispensable role of D1 receptor in aversive pavlovian conditioning. Additionally, pharmacological studies reveal the significant role of D1-type receptors in appetitive pavlovian conditioning in mammals (Schroeder, 2000; Baker, 2003; Eyny, 2003; Dalley, 2005) and possibly in Aplysia (Reyes, 2005), although no information is available on D1 or D5 knock-out mice in this type of behavioral plasticity. Therefore, the studies described here elucidate, for the first time, the critical role of D1 receptor in appetitive pavlovian conditioning (Kim, 2007b).

Dopamine receptor DAMB signals via Gq to mediate forgetting in Drosophila

Prior studies have shown that aversive olfactory memory is acquired by dopamine acting on a specific receptor, dDA1, expressed by mushroom body neurons. Active forgetting is mediated by dopamine acting on another receptor, Damb, expressed by the same neurons. Surprisingly, prior studies have shown that both receptors stimulate cyclic AMP (cAMP) accumulation, presenting an enigma of how mushroom body neurons distinguish between acquisition and forgetting signals. This study surveyed the spectrum of G protein coupling of dDA1 and Damb, and it was confirmed that both receptors can couple to Gs to stimulate cAMP synthesis. However, the Damb receptor uniquely activates Gq to mobilize Ca(2+) signaling with greater efficiency and dopamine sensitivity. The knockdown of Galphaq with RNAi in the mushroom bodies inhibits forgetting but has no effect on acquisition. These findings identify a Damb/Gq-signaling pathway that stimulates forgetting and resolves the opposing effects of dopamine on acquisition and forgetting (Himmelreich, 2017).

This study provides biochemical and behavioral evidence that the Drosophila DA receptor Damb couples preferentially to Gαq to mediate signaling by Damb for active forgetting. This conclusion offers an interesting contrast to the role of the dDA1 receptor in MBns for acquisition, and it resolves the issue of how MBns distinguish DA-mediated instructions to acquire memory versus those to forget. Prior studies had classified both dDA1 and Damb as cAMP-stimulating receptors, similar to mammalian D1/D5 DA receptors that work through Gαs/olf. The results extend prior studies of dDA1 by examining coupling of this receptor with multiple heterotrimeric G proteins to show that the receptor strongly and preferentially couples to Gs proteins. This affirms the receptor's role in the acquisition of memory, consistent with the tight link between acquisition and cAMP signaling. This study found that the Damb receptor can weakly couple to Gs proteins but preferentially engages Gq to trigger the Ca2+-signaling pathway, a feature not displayed by dDA1. Comparing the two Gαq paralogs of Drosophila (G and D) with a human ortholog shows that Drosophila GαqG and human Gαq share a conserved C terminus, essential for selective coupling to GPCRs, but quite distinct in sequence compared to the GαqD C terminus. Since GαqD is a photoreceptor-selective G protein that couples with rhodopsin, it is proposed that GαqG is the isoform that relays Damb's signals to spur forgetting (Himmelreich, 2017).

It is envisioned that memory acquisition triggered by strong DA release from electric shock pulses used for aversive conditioning drives both cAMP and Ca2+ signaling through dDA1 and Damb receptors in the MBns. Forgetting occurs from weaker DA release after the acquisition through restricted Damb/Gαq/Ca2+ signaling in the MBns. The coupling of Damb to Gs at high DA concentrations also explains why Damb mutants have a slight acquisition defect after training with a large number of shocks. Although the model allows the assignment of acquisition and forgetting to two distinct intracellular signaling pathways, it does not preclude the possibility that other differences in signaling distinguish acquisition from forgetting. These include the possible use of different presynaptic signals, such as a co-neurotransmitter released only during acquisition or forgetting (Himmelreich, 2017).

Octopamine and Dopamine differentially modulate the nicotine-induced calcium response in Drosophila Mushroom Body Kenyon Cells

In Drosophila associative olfactory learning, an odor, the conditioned stimulus (CS), is paired to an unconditioned stimulus (US). The CS and US information arrive at the Mushroom Bodies (MB), a Drosophila brain region that processes the information to generate new memories. It has been shown that olfactory information is conveyed through cholinergic inputs that activate nicotinic acetylcholine receptors (nAChRs) in the MB, while the US is coded by biogenic amine (BA) systems that innervate the MB. In this regard, the MB acts as a coincidence detector. A better understanding of the properties of the responses gated by nicotinic and BA receptors are required to get insights on the cellular and molecular mechanisms responsible for memory formation. In recent years, information has become available on the properties of the responses induced by nAChR activation in Kenyon Cells (KCs), the main neuronal MB population. However, very little information exists on the responses induced by aminergic systems in fly MB. This study evaluated some of the properties of the calcium responses gated by Dopamine (DA) and Octopamine (Oct) in identified KCs in culture. Exposure to BAs induces a fast but rather modest increase in intracellular calcium levels in cultured KCs. The responses to Oct and DA are fully blocked by a Voltage-gated Calcium Channel (VGCC) blocker, while they are differentially modulated by cAMP. Moreover, co-application of BAs and nicotine has different effects on intracellular calcium levels: while DA and nicotine effects are additive, Oct and nicotine induce a synergistic increase in calcium levels. These results suggest that a differential modulation of nicotine-induced calcium increase by DA and Oct could contribute to the events leading to learning and memory in flies (Leyton, 2013).

Two DA receptors that share homology to vertebrate DA type 1 receptors are expressed in Drosophila MB, DAMB (Dopamine 1-like receptor 2 or Dop1R2) and dDA1/DmDOP1/DopR. These receptors have been cloned and expressed in heterologous systems, where they are positively coupled to AC. Moreover, these receptors participate in the generation or modification of new olfactory memories in MB. The third cloned DA receptor, Dop2R/D2R, is not expressed in Drosophila MB. The current results are consistent with the general idea that DA receptors modulate intracellular cAMP levels, which could lead to the modification of the activity of VGCCs in KCs. The EC50 describe in this study is in agreement with previous studies in heterologous systems, which report EC50 in the range of 300-500 nM for both DA type 1 receptors. Thus, it is very likely that DAMB and/or dDA1/DmDOP1 are contributing to the DA-induced calcium response in Drosophila KCs. It would be expected that DA receptors increase cAMP levels to activate calcium currents in fly MB KCs. However, data using SQ22536 suggest that cAMP inhibits calcium fluxes in KCs. Although the cellular mechanisms responsible for this effect are not evident, it has been previously shown that increased cAMP signaling negatively modulates the activity of VGCCs through the activation of specific phosphatases in the vertebrate Nucleus Accumbens. Remarkably, the Nucleus Accumbens and MB are brain regions highly associated to the plastic behavioral effects induced by addictive drugs. Thus, it would not be particularly surprising to find similarities in the mechanisms responsible for the modulation of neuronal communication and excitability byDA in these two brain structures, as previously suggested.On the other hand, several Oct receptors have been previously cloned in Drosophila: one Oct receptor with high sequence homology to vertebrate -type receptors (Oct1R/OAMB) is expressed in dendrites and axons of the MB, and is the main candidate for the calcium responses induced by Oct in KCs, since the other cloned Oct receptors are not expressed in MB. In agreement with this, the calculated EC50, and the description that the response depends on VGCC activation and is independent on cAMP, further support this suggestion (Leyton, 2013).

An interaction of the neural systems responsible for CS and US stimuli in the MB region could mediate the generation of new memories. This interaction could occur at the presynaptic level, for instance, through the nAChR modulation of aminergic innervation to the MB region. However, the most accepted idea is that cholinergic and aminergic receptors expressed in MB KCs gate intracellular cascades that could cross-talk to modify the activity of KCs, a cellular event that could underlie long-lasting changes responsible for the generation of new olfactory memories in the fly (Leyton, 2013).

Although the modulation of nAChR-elicited responses by BAs in MB KCs is not a new idea in the field, direct evidence to support this proposition is limited. Previous studies have shown that DA modifies the acetylcholine-induced calcium response detected in MB in an in vivo fly brain preparation. That data show that this modulation depends on cAMP signaling. In addition to this, it has been shown that the enhancement in synaptic transmission between antennal lobe neurons and MB KCs in vitro depends on the activation of different receptors including dDA1/DmDOP1 and nAChRs. All these data support the idea that dopaminergic systems modulate the properties of the cholinergic responses in KCs. The proposition that Oct modulates nicotinic responses has not been previously evaluated. It is an interesting observation that the amines differentially modulate the calcium increase observed in presence of nicotine: while Oct and nicotine induce a synergistic effect on intracellular calcium levels compared to the responses detected for each ligand, the effects of DA and nicotine are rather additive. It is also interesting that this synergistic event is only observed when KCs are exposed to nicotine in presence of a saturating concentration of Oct, and is not observed at a lower Oct concentration (Leyton, 2013).

Neural control of startle-induced locomotion by the mushroom bodies and associated neurons in Drosophila

Startle-induced locomotion is commonly used in Drosophila research to monitor locomotor reactivity and its progressive decline with age or under various neuropathological conditions. A widely used paradigm is startle-induced negative geotaxis (SING), in which flies entrapped in a narrow column react to a gentle mechanical shock by climbing rapidly upwards. This study combined in vivo manipulation of neuronal activity and splitGFP reconstitution across cells to search for brain neurons and putative circuits that regulate this behavior. The activity of specific clusters of dopaminergic neurons (DANs) afferent to the mushroom bodies (MBs) modulates SING, and DAN-mediated SING regulation requires expression of the DA receptor Dop1R1/Dumb, but not Dop1R2/Damb, in intrinsic MB Kenyon cells (KCs). Previous observations were confirmed that activating the MB α'β', but not αβ, KCs decreased the SING response, and further MB neurons implicated in SING control were identified, including KCs of the γ lobe and two subtypes of MB output neurons (MBONs). Co-activating the αβ KCs antagonizes α'β' and γ KC-mediated SING modulation, suggesting the existence of subtle regulation mechanisms between the different MB lobes in locomotion control. Overall, this study contributes to an emerging picture of the brain circuits modulating locomotor reactivity in Drosophila that appear both to overlap and differ from those underlying associative learning and memory, sleep/wake state and stress-induced hyperactivity (Sun, 2018).

This study has identified MB afferent, intrinsic and efferent neurons that underlie modulation of startle-induced locomotion in the Drosophila brain. Using in vivo activation or silencing of synaptic transmission in neuronal subsets, specific compartments of the MBs were shown to be central to this modulation. Implicated neurons include α'β' and γ KCs, subsets of PAM and PPL1 DANs, and the MBONs V2 and M4/M6. Some of the potential synaptic connections between these elements were characterized using splitGFP reconstitution across cells. Although the picture is not complete, these results led to a proposal of a scheme of the neuronal circuits underlying the control of locomotor reactivity in an insect brain (Sun, 2018).

It has been previously reported that the degeneration of DANs afferent to the MBs in the PAM and PPL1 clusters is associated with accelerated decline of SING performance in aging flies. This study has specifically addressed the role of these and other DANs in SING modulation. The initial observation was that thermoactivation of TH-Gal4-targeted DANs consistently led to decreased locomotor reactivity, while silencing synaptic output from these neurons had no effect. This result was verified by rapid optogenetic photostimulation, indicating that indeed DAN activation affects locomotor reactivity during the execution of the behavior. In contrast, blocking selectively synaptic output of the PAM DANs neurons resulted in a slight increase in SING performance, suggesting that a subset of spontaneously active neurons in the PAM inhibits SING. It should be noted, however, that this effect appeared small probably in part because SING performance was already very high for the control flies in the assay condition. This issue may have prevented detection of other modulatory neurons in the course of this study. Interestingly, the data suggest that those PAM neurons that inhibit SING are targeted by NP6510-Gal4, a driver that expresses in 15 PAM DANs that project to the MB β1 and β'2 compartments. The degeneration of these neurons also appears to be largely responsible for α-synuclein-induced decline in SING performance in a Parkinson disease model. Moreover, one observation is provided in this study, using DAN co-activation with TH-Gal4 and R58E02-Gal4, suggesting that other subsets of the PAM cluster may modulate locomotor reactivity with opposite effects, i.e., increase SING when they are stimulated (Sun, 2018).

This study further indicated that thermoactivation of two DANs of the PPL1 cluster, either MB-MP1 that projects to the γ1 peduncle in the MB horizontal lobes or MB-V1 that projects to the α2 and α'2 compartments of the MB vertical lobes, was sufficient to significantly decrease SING performance. This suggests that the MB-afferent DANs of the PPL1 cluster are also implicated in SING modulation. Other DAN subsets could play a role and are still to be identified. However, inactivation of a DA receptor, Dop1R1/Dumb, in MB KCs precluded DAN-mediated SING modulation, strongly suggesting that DANs afferent to the MBs play a prominent role in the neuronal network controlling fly's locomotor reactivity. In contrast, inactivating Dop1R2/Dumb in KCs did not show any effect on DA-induced SING control (Sun, 2018).

Therefore, these results suggest that DA input to the MBs can inhibit or increase the reflexive locomotor response to a mechanical startle, allowing the animal to react to an instant, sudden stimulus. In accordance with this interpretation, previous reports have shown that the MB is not only a site for associative olfactory learning, but that it can also regulate innate behaviors. By combining synaptic imaging and electrophysiology, a previous study demonstrated that dopaminergic inputs to the MB intrinsic KCs play a central role in this function by exquisitely modulating the synapses that control MB output activity, thereby enabling the activation of different behavioral circuits according to contextual cues (Sun, 2018).

A decrease in SING performance has been previously reported when KCs in the α'β' lobes, but not in the αβ and γ lobes, were thermogenetically stimulated or their synaptic output silenced. Using a set of specific drivers, the contribution of the various MB lobes in the modulation of this innate reflex was precisely studied. It was confirmed that the α'β' KCs down-regulate SING when they are activated but not when their output is inhibited. Other unidentified neurons, targeted by the rather non-selective c305a-Gal4 and G0050-Gal4 drivers, trigger a decrease in SING performance when they are inhibited by Shits1, and are therefore potential SING-activating neurons. It was further found that the MB γ lobes contain KCs that strongly inhibit SING when activated, both by thermogenetic and optogenetic stimulation, as shown with the γ-lobe specific driver R16A06-Gal4. However, thermoactivation of γ neurons with other drivers, like mb247-Gal4, which express both in the αβ and γ lobe, did not decrease SING. This could result from an inhibitory effect of αβ neuron activation on SING modulation by γ neurons. To test this hypothesis, a double-driver was generated by recombining mb247-Gal4 with R16A06-Gal4. Because both drivers express in the γ lobes, one would expect a stronger effect on SING modulation after thermoactivation with the double-driver than with R16A06-Gal4 alone. The opposite was observed, i.e., that SING was decreased to a lesser extent with the double-driver than with R16A06-Gal4 alone. Activation of mb247-Gal4 αβ neurons therefore likely counterbalanced the effect of γ neuron activation with R16A06-Gal4 on SING modulation. A similar and even more obvious result was obtained when mb247-Gal4 was recombined with the α'β' driver R35B12-Gal4: co-activation of the neurons targeted by these two drivers prevented the strong SING modulation normally induced by R35B12-Gal4 alone. These results suggest the existence of an inter-compartmental communication process for locomotor reactivity control in the Drosophila MB. Comparably, it was recently suggested, in the case of memory retrieval, that MB output channels are ultimately pooled such that blockade (or activation) of all the outputs from a given population of KCs may have no apparent effect on odor-driven behavior, while such behavior can be changed by blocking a single output. Such a transfer of information could occur, as was previously reported, through connections involving the MBONs within the lobes or outside the MB (Sun, 2018).

Finally, the activation of two sets of MB efferent neurons, cholinergic MBON-V2 and glutamatergic MBON-M4/M6, consistently decreased SING performance of the flies. In contrast, silencing these neurons had no effect on locomotor behavior. The dendrites of these MBONs arborize in the medial part of the vertical lobes (α2, α'3) and the tips of the horizontal lobes (β'2 and γ5), respectively, as further evidence that the prime and γ lobes, and DANs efferent to these compartments, are involved in SING modulation. Results are also shown from GRASP observations suggesting that the PAM DANs either lay very close or in some other manner make potential synaptic connections with the MBON-M4/M6 neurons in their MB compartments, as well as the M4/M6 with the PAM in the SMP. The results also provide evidence that the PPL1 DANs and MBON-V2 contact each other in the vertical lobes and that axo-axonic synaptic contacts may occur between the MBON-V2 and M4/M6 neurons in their common projection region in the SMP (Sun, 2018).

These MBONs are known to be involved in opposite ways in olfactory memory: DAN-induced synaptic repression of cholinergic or glutamatergic MBONs would result in the expression of aversive or attractive memory, respectively. This study finds, in contrast, that the activation of these two sets of MBONs had similar depressing effects on SING behavior. Interestingly, it has been recently reported that the glutamatergic MBONs and PAM neurons that project to the MB β'2 compartment are also required for modulation of another innate reflex, CO2 avoidance (Lewis, 2015). CO2 exposure, like mechanical startle, represents a potential danger for the flies, thus triggering an avoidance behavior that can be suppressed by silencing these MBONs in specific environmental conditions. However, it is the activation of glutamatergic MBONs that inhibits SING. This apparent discrepancy might be explained if the downstream circuits were different for these two escape behaviors (CO2 avoidance and fast climbing). Overall, the current results further support the hypothesis of a primary role of the MB as a higher brain center for adapting innate sensory-driven reflex to a specific behavioral context (Cohn, 2015; Lewis, 2015; Sun, 2018 and references therein).

Even though the model remains to be confirmed and elaborated, a proposed scheme summarizing the current working hypothesis is presented of the neural components underlying SING control. Sensory information from mechanical stimulation triggers an innate climbing reflex (negative geotaxis) that can be regulated by signals transmitted from MB-afferent DANs (in the PAM and PPL1 cluster) to select KCs and two sets of MBONs (V2 and M4/M6) in specific MB compartments. Processing of this information could occur through synergistic or antagonistic interactions between the MB compartments and, finally, the MBON neurons would convey the resulting modulatory signal to downstream motor circuits controlling the climbing reflex. It was observed that the axonal projections of these MBONs make synaptic contacts with each other and converge together to the SMP where the dendrites of DANs lie, suggesting that these projections might form feedback loops to control DA signaling in the circuits (Sun, 2018).

DA signals for SING modulation originate from PAM neuron subsets and neurons inside the PPL1 cluster (MB-MP1 and MB-V1) that project to the MB lobes (see Schematic representation of MB-associated neural components modulating startle-induced locomotion). Axon of MB-V1 is shown as a dashed line because a driver specific for this neuron could not be tested in this study. The α'β' and γ KCs appear to be the main information integration center in this network, while their effect on SING modulation is opposed by the activity of αβ lobe KCs. Processed SING modulation signals are then transferred by two subtypes of MB efferent neurons, MBON-V2 and M4/M6, to the downstream SING reflex motor circuits. These two MBON subtypes have their axons converging together in the SMP where they may form axo-axonic synaptic connections, in which MBON-V2 would be presynaptic to MBON-M4/M6. The SMP also contains dendrites of the PAM and PPL1 DANs, thereby potentially forming instructive feedback loops on DA-mediated SING modulation. Most neurons identified here downregulated SING performance when they were activated, except for a subset of the PAM clusters that appeared constitutively inhibitory (represented as darker neurons in the figure) and the αβ lobe KCs that seem to antagonize SING modulation by other MB neurons. The different MB lobes are shown in various shades of green as indicated. The PAM DANs, PPL1 DANs and MBONs are drawn in magenta, light blue and dark gray, respectively. PAM: protocerebral anterior medial; PPL1: protocerebral posterior lateral; MBON: mushroom body output neuron; SMP superior medial protocerebrum; ped: peduncle; pre: presynaptic; pos: postsynaptic (Sun, 2018).

SING performance can be affected by a collection of factors including the arousal threshold of the fly, the ability to sense gravity and also climbing ability. 'Arousal' is defined as a state characterized by increased motor activity, sensitivity to sensory stimuli, and certain patterns of brain activity. A distinction can be made between endogenous arousal (i.e., wakefulness as opposed to sleep) and exogenous arousal (i.e., behavioral responsiveness). In Drosophila, DA level and signaling control all known forms of arousal. Because the MB plays an important role in sleep regulation, sleep- or wake-promoting networks might indeed in part interact or overlap with those controlling locomotor reactivity. However, this study observed that thermoactivation with various drivers had in a number of cases opposite effects on sleep/wake state and SING. First, neuronal thermoactivation with TH-Gal4 suppresses sleep but decreases the SING response. Second, extensive thermogenetic activation screen revealed that α′β′ and γm KCs are wake-promoting and γd KCs are sleep-promoting. In the current experiments, neuronal activation of α′β′ or γ KCs both led to strongly decreased locomotor reactivity. Third, stimulating MBON-M4 and M6, which are wake-promoting, decreased SING performance (Sun, 2018).

Another brain structure, the EB, plays important roles in the control of locomotor patterns and is also sleep-promoting. Furthermore, the EB is involved in the dopaminergic control of stress- or ethanol-induced hyperactivity, which can be considered as forms of exogenously-generated arousal. Several drivers labeling diverse EB neuronal layers were used, and no noticeable effects of thermoactivation of these neurons on the SING response was found. It is concluded that the circuits responsible for SING modulation, although they apparently share some similarities, are globally different from those controlling sleep/wake state and environmentally-induced hyperactivity (Sun, 2018).

Overall, this work identified elements of the neuronal networks controlling startle-induced locomotion in Drosophila and confirmed the central role of the MBs in this important function. Future studies are required to complete this scheme and explore the intriguing interactions between the different MB compartments in SING neuromodulation (Sun, 2018).

Distinct dopamine receptor pathways underlie the temporal sensitivity of associative learning

Animals rely on the relative timing of events in their environment to form and update predictive associations, but the molecular and circuit mechanisms for this temporal sensitivity remain incompletely understood. This study shows that olfactory associations in Drosophila can be written and reversed on a trial-by-trial basis depending on the temporal relationship between an odor cue and dopaminergic reinforcement. Through the synchronous recording of neural activity and behavior, this study shows that reversals in learned odor attraction correlate with bidirectional neural plasticity in the mushroom body, the associative olfactory center of the fly. Two dopamine receptors, DopR1 and DopR2, contribute to this temporal sensitivity by coupling to distinct second messengers and directing either synaptic depression or potentiation. These results reveal how dopamine-receptor signaling pathways can detect the order of events to instruct opposing forms of synaptic and behavioral plasticity, allowing animals to flexibly update their associations in a dynamic environment (Handler, 2019).

While memories are often thought of as windows into the past, their adaptive value lies in the ability to predict the future. This study, took advantage of the concise circuitry of the Drosophila mushroom body to investigate how the precise timing of dopaminergic reinforcement allows animals to form and maintain predictive associations between cues and outcomes. While studies of associative learning have often focused on sensory cues that anticipate punishment or reward, equally informative are cues associated with their termination. This study demonstrates that shifting the relative timing of an odor and reinforcement by <1 s can switch the valence of an olfactory memory, underscoring the exquisite temporal sensitivity of this circuit. As a consequence, flies can form equivalent appetitive associations with odors that anticipate rewards or follow punishments, or aversive associations with odors that predict punishments or follow rewards. The symmetry of this behavioral modulation permits Drosophila to take advantage of all the temporally correlated features of their environment that can be used to infer causal relationships. Together, this work suggests a model in which the steep temporal sensitivity of associative learning arises from the concerted action of two dopamine receptor-signaling pathways that work in opposition to bidirectionally regulate the strength of KC-MBON signaling (see DopR1 and DopR2 Are Required for Behavioral Flexibility), allowing animals to maintain an accurate model of a complex and changing world (Handler, 2019).

In a dynamic environment, memories must be continually retouched and rewritten to maintain their relevance and predictive value. By monitoring how individual flies adapt their odor preferences over 50 conditioning trials, this study has revealed that Drosophila can form and reverse learned associations on a trial-by-trial basis, pointing to the fundamental flexibility of memory updating mechanisms (Handler, 2019).

Prior work in both Drosophila and mammals has suggested memory retention is regulated by multiple mechanisms at different timescales. If not reinforced, memories may passively fade over time, reflecting the natural turnover of molecular and neural hardware. Alternatively, memories can be actively eroded either by re-exposure to the learned odor in the absence of the anticipated dopaminergic reinforcement or the reinforcement in the absence of the odor, violating the expected contingency between these two events. In contrast, the brief episodes of odor and dopaminergic reinforcement (1-2 s) used in the current study are insufficient to overwrite an olfactory association when presented independently but can immediately reverse a prior association when paired together in time. The convergence of olfactory and DAN input to the mushroom body thus conveys information about their causal relationship, offering a mechanism to rapidly update memories to reflect the changing temporal structure of the environment (Handler, 2019).

While memory updating could rely on plasticity at various sites within this circuit, this study demonstrated that the bidirectional modulation of behavior is highly correlated with bidirectional changes in the strength of the same KC-MBON synapses within the mushroom body. Such bidirectional synaptic plasticity has been proposed to confer reversibility to learning circuits. For example, spike-timing dependent plasticity (STDP) can bidirectionally tune the strength of synaptic connections between neurons depending on the relative timing of spikes in pre- and post-synaptic neurons, mirroring the sensitivity to temporal order observed in associative learning. However, STDP requires nearly coincident firing patterns on a millisecond timescale, far more rapid than the temporal relationships between stimuli typically required for associative learning. In this study, by examining neural and behavioral modulation over the same timescales and even concurrently within the same individuals, the modulation of synaptic signaling within the mushroom body is linked to reversible changes in behavior (Handler, 2019).

Within the mushroom body, each compartment serves as a site of convergence between odor signaling conveyed by KCs and dopaminergic reinforcement, allowing dopamine-receptor pathways within KC axons to detect the temporal order of these inputs. The spatial patterns of dopamine release and dopamine receptor second-messenger cascades are found to adhere to the compartmentalized architecture of the lobes, permitting different synapses along the same KC axon to be independently regulated. These observations suggest that within a compartment, multiple neuromodulatory mechanisms tune neurotransmission depending on the temporal structure of conditioning. As a consequence, the distinct complement of KC-MBON synapses activated by odors that precede or follow a reinforcement are differentially regulated, allowing a single dopaminergic reinforcement to drive the synchronous formation of multiple odor associations, effectively enhancing the coding capacity of a compartment (Handler, 2019).

Dopamine shapes circuit function in diverse ways by engaging distinct classes of receptors that couple to different signaling cascades. In Drosophila, DopR1 and DopR2 have been proposed to play opposing roles in olfactory memory regulation at the behavioral level, with DopR1 essential to memory formation and DopR2 necessary for memory erosion. Yet the contribution of these receptors to synaptic modulation within the mushroom body has remained unclear. The current work reveals that the opposing behavioral roles of DopR1 and DopR2 are mirrored by their antagonistic regulation of KC-MBON signaling, with DopR1 required for the depression ensuing from forward pairing, while DopR2 is essential for the potentiation that follows backward pairing. Although DANs selectively innervating the mushroom body are sufficient to instruct bidirectional behavioral modulation, the broader expression of DopR1 and DopR2 leaves open the possibility that these receptors may also act at other sites within the nervous system to shape the temporal sensitivity of associative learning (Handler, 2019).

Using fluorescent sensors of DopR1 and DopR2 second messengers allowed the gaining insight into the spatial and temporal patterning of these intracellular signaling pathways during conditioning. While a potential limitation of optical reporters is their restricted sensitivity and dynamic range, these sensors nevertheless reveal that the selective recruitment of dopamine-receptor signaling cascades is sufficient to account for the temporal dependence of neural and behavioral modulation. Monitoring cAMP production during conditioning reveals that, while the DopR1 pathway serves as a coincidence detector, in accord with the coordinate regulation of adenylate cyclases by Gαs and calcium, it cannot autonomously encode the temporal order of events. In contrast, DopR2 signaling through Gαq strictly depends on the temporal sequence of KC and DAN activation (Handler, 2019).

Which component of the DopR2-signaling cascade is sensitive to the temporal ordering of odor and reinforcement? IP3 receptors that gate calcium release from the ER lumen represent an intriguing candidate, as their complex regulation by both IP3 and cytosolic calcium renders them inherently sensitive to the sequence of agonist binding: IP3 binding unmasks a calcium regulatory site required for channel opening, while high calcium in the absence of IP3 inhibits channel activity. Indeed, this study observed that ER calcium release is time locked to KC stimulation suggesting that the precise order dependence of this pathway relies on calcium entry subsequent to IP3 production. In the cerebellum, bidirectional plasticity at parallel fiber-Purkinje neuron synapses has been proposed to similarly rely on calcium release from the ER lumen via IP3 receptors. While the analogous circuit organization of the mushroom body and cerebellum has been well described, the current observations suggest they may share conserved molecular mechanisms for temporally precise synaptic modulation (Handler, 2019).

Together, this work points to dopamine receptor signaling pathways in KC axons as a key site of temporal coincidence and order detection during associative learning. While this study focused on the role of dopaminergic signaling within the γ4 compartment, timing-dependent bidirectional plasticity was observed to be shared characteristic of KC-MBON synapses in multiple compartments of the γ lobe. Therefore, the reversible modulation of behavior instructed by both the PAM or PPL DANs likely reflects bidirectional plasticity driven synchronously in the multiple compartments innervated by these DAN drivers. Aversive electric shock and sugar rewards evoke distributed patterns of activity across the DAN population, implying that these naturalistic reinforcers likewise instruct coordinated bidirectional plasticity across different compartments to rapidly shape the net output of the mushroom body. Similar patterns of DAN activity are also elicited by a fly's locomotion, raising the possibility that, in the context of an odor plume, an animal's behavior may serve as a reinforcement stimulus that itself drives bidirectional synaptic plasticity to regulate odor processing (Handler, 2019).

The ability to form or overwrite associations on a trial-by-trial basis allows for adaptive behavior in a noisy and uncertain environment where the temporal relationships between events may quickly change. However, animals must also have the capacity to store relevant memories persistently, even for a lifetime. Therefore, the reversible plasticity observed must co-exist with additional molecular and circuit mechanisms that underlie the formation and retention of longer-term associations. Indeed, recent work has described intrinsic differences between mushroom body compartments in their susceptibility to memory erosion as well as differences in second-messenger signaling in distinct KC subpopulations. Together, these results suggest that the differential expression or coupling of dopamine-receptor signaling pathways in different KC classes may tune synaptic plasticity rules to regulate the persistence of information storage. While this work connects molecular pathways within a sub-population of KCs to the emergence of short-term associations, functional dissection of these signaling cascades across the different lobes of the mushroom body will provide insight into the distinct timescales of memory formation and erosion (Handler, 2019).

Compartment specific regulation of sleep by mushroom body requires GABA and dopaminergic signaling
Sleep is a fundamental behavioral state important for survival and is universal in animals with sufficiently complex nervous systems. As a highly conserved neurobehavioral state, sleep has been described in species ranging from jellyfish to humans. Biogenic amines like dopamine, serotonin and norepinephrine have been shown to be critical for sleep regulation across species but the precise circuit mechanisms underlying how amines control persistence of sleep, arousal and wakefulness remain unclear. The fruit fly, Drosophila melanogaster, provides a powerful model system for the study of sleep and circuit mechanisms underlying state transitions and persistence of states to meet the organisms motivational and cognitive needs. In Drosophila, two neuropils in the central brain, the mushroom body (MB) and the central complex (CX) have been shown to influence sleep homeostasis and receive aminergic neuromodulator input critical to sleep-wake switch. Dopamine neurons (DANs) are prevalent neuromodulator inputs to the MB but the mechanisms by which they interact with and regulate sleep- and wake-promoting neurons within MB are unknown. This study investigated the role of subsets of PAM-DANs that signal wakefulness and project to wake-promoting compartments of the MB. PAM-DANs were found to be GABA responsive and required GABA(A)-Rdl receptor in regulating sleep. In mapping the pathways downstream of PAM (protocerebral anterior medial) neurons innervating γ5 and β'2 MB compartments it was found that wakefulness is regulated by both DopR1 and DopR2 receptors in downstream Kenyon cells (KCs) and mushroom body output neurons (MBONs). Taken together, this study has identified and characterized a dopamine modulated sleep microcircuit within the mushroom body that has previously been shown to convey information about positive and negative valence critical for memory formation. These studies will pave way for understanding how flies balance sleep, wakefulness and arousal (Driscoll, 2021).

The mushroom body lobes are tiled by discrete anatomic compartments defined by the axons of a specific subset of DANs and the dendrites of one or two mushroom body output neurons (MBONs). This anatomical arrangement positions DANs to strategically convey positive and negative reinforced information by changing the synaptic weight of KC-MBONs in producing aversive and appetitive responses (Driscoll, 2021).

While, the most in-depth analysis of these synapses and distinct DAN-KC-MBON connectivity and behavioral output comes from studies of olfactory conditioning, there is evidence that these synapses play a critical role in innate behaviors like feeding and sleep. Although, role of DA on sleep has been extensively investigated in Drosophila, the commonly used TH-Gal4 driver line labels most dopamine neuron clusters, but is absent from the several PAM clusters that projects to MB (Driscoll, 2021).

This study specifically probed PAM subsets that project to γ5, γ4, and β'2 MB compartments. This study focused on this subset because KCs and MBONs downstream of these PAM neurons can be neuroanatomically resolved and have been shown to be required for wakefulness. Further, KCs and MBONs that form the γ5, γ4, and β'2 synaptic compartments alter their spontaneous neural activity in response to sleep need (induced by mechanical sleep-deprivation). The ability to use cell-specific split-GAL4 tools provides opportunity to resolve the precise circuit mechanisms by which PAM neurons regulate wakefulness (Driscoll, 2021).

GABA signaling also modulates sleep and wake microcircuits within MB. The key source of GABA in the MB is anterior paired lateral neurons, APL and dorsal paired medial neurons (DPM), which are electrically coupled and increase sleep by GABAergic inhibition of wake-promoting KCs. In the context of associative learning, there is strong evidence for interactions between KCs, APL, DPM and DANs but it is not clear if GABA and dopamine signaling represent opposing inputs to the KCs and MBONs in the regulation of sleep. This study found that the excitability of PAM DANs involved in wakefulness is blocked by sleep-promoting GABA signaling and mediated by ionotropic receptor subtype GABAA-Rdl (Driscoll, 2021).

A recent study showed that GABA inhibitory input to the presynaptic terminals of the PAM neurons regulates appetitive memory and that this interaction is mediated by GABA-B3 receptors that are clustered in PAM boutons localized to PAM-γ5 and -α1 compartments. These data are consistent with the findings that PAM-γ5 are GABA responsive and that multiple receptors are critical to this interaction. Since, no role was found for GABA-B3 in PAM mediated sleep regulation, it is likely that PAM γ5, γ4, and β'2 express multiple GABA receptors which are differentially recruited in sleep and learning. How and what regulates the expression of these receptors in PAM subsets presents a potential mechanism of presynaptic gating to MB core circuits. Transcriptomic analysis of PAM neurons reveals extremely high levels of Rdl expression followed by GABA-B3. Among the PAM subsets mean TPM or transcripts per million of Rdl receptor in PAM γ5, γ4, and β'2 are much higher as compared to other PAM subsets (Driscoll, 2021).

Simple connection query search of the recently released hemibrain data85 reveals there is significant bidirectional connectivity between APL, DPM, and PAM neurons. Further, a recent study showed that APL neurons express the inhibitory D2R receptor55. APL mediated GABAergic inhibition of the PAM neurons was recently shown to control the intensity and specificity of olfactory appetitive memory but previous results show that blocking GABA release from APL neurons only modestly affects sleep phenotypes (Driscoll, 2021).

While, the role of APL in GABA signaling to PAM γ5, γ4, and β'2 cannot be completely ruled out, other inputs to wake-regulating PAM DANs could also be GABAergic and critical for promoting sleep. A recent study using EM dataset of a Full Adult Female Fly Brain (FAFB) mapped the inputs and outputs of the PAMγ5 DANs and identified that this cell type is highly heterogenous and in addition to recurrent feedback from MBON01 γ5β'2a, it receives extensive input from other MBONs, sub-esophageal output neurons (SEZONs) and lateral horn output neurons86. The EM data also reveals that octopaminergic neurons synapse onto PAM γ5, γ4, and β'2 DANs. Whether, these inputs play a role in wakefulness is unknown but suggests that the PAMγ5 could serve as a key link between sensory inputs, wake-promoting octopamine signal and core sleep regulating circuitry within the MB. Each of these inputs could modulate PAM-DAN activity and dopamine release in regulating wakefulness via the MB (Driscoll, 2021).

In addition to probing the release and activity of these PAM-DANs the dopamine receptors and their location within the MB in signaling wakefulness were also explored. To this end validated RNAi lines were expressed in subsets of KCs and MBONs; DopR1 and DopR2 were found to be critical in mediating the wakefulness signal via KCs and γ5β'2 MBONs. Knocking down the receptor consistently increased total sleep and bout length. Furthermore, specific manipulations of DopR receptors within the MB did not directly alter locomotor activity as observed by manipulation of these receptors in CX. Although, loss-of-function mutations of D1 dopamine receptor DopR are shown to enhance repetitive air puff startle-induced arousal and increase sleep. Expression and restoration of DopR in the mutant background specifically in the central complex rescues the startle response, while, the sleep phenotype is rescued via a broad MB driver. The current data extends these findings by showing that the DopR receptors regulate sleep via the MB γ5 and β'2 compartment. Although, targeted RNAi experiments show that DopR's are required for sleep regulation by KCs and MBONs, the lack of a sleep phenotype in DopR2 mutant could be a result of global loss of receptor in the mutant as opposed to targeted loss of receptor function within MB. Dopamine signals wakefulness by activation of wake-promoting neurons of MB via DopR1 and DopR2 and within. the central complex, neurons of dFB are inhibited by dopamine via DopR2. Hence, DopR2 has opposing effects within MB and CX (Driscoll, 2021).

In vitro characterization indicates that DopR's signal through distinct G-proteins, with DopR1 via Gαs to stimulate cAMP production and DopR2 coupling to Gαq via increased calcium. These receptors are thought to have differential sensitivity to dopamine and could be potentially recruited by varying DA release or DAN activity. In the context of sleep regulation, this work reveals that both DopR1 and DopR2 induce wakefulness via the γ5 β'2 MB compartment but not γ4 compartment. Although, chronic activation of PAM γ4 induces wakefulness, the glutamatergic MBON γ4 < γ1,2 projects to multiple compartments and could potentially activate or inhibit MBONs and PAMs projecting to γ1 and γ2 compartment. The interaction between compartments is not well understood in the context of sleep and wake regulation and requires further investigation to better understand the role of DopR2 in regulating the γ4 compartment. The neuroanatomical specificity obtained from split-Gal4 lines combined with EM data has paved way for more detailed analysis of the role of dopamine signaling to MB in the context of sleep and other behaviors (Driscoll, 2021).

The sleep-regulating PAM DANs and associated KCs and MBONs identified in this study are also involved in mediating satiety, novelty, caffeine induced arousal, punishment and reward associated experiences suggesting that the activity of these neurons is tuned to several wake and arousal associated behaviors. This is further supported by the EM connectome data showing that MB receives extensive gustatory, auditory and visual input in addition to olfactory input (Driscoll, 2021).

Current models of sleep regulation rely on two main processes, the circadian clock and the sleep homeostat and don't completely account for multiple external and internal factors that influence wakefulness. The ability to sleep, however, is influenced by motivational or cognitive stimuli. It is therefore envisioned that sleep, wakefulness and arousal within MB are not located in distinct circuits, but rather mediated by distinct processes within a common circuit (Driscoll, 2021).

Innate and learned odor-guided behaviors utilize distinct molecular signaling pathways in a shared dopaminergic circuit

Odor-based learning and innate odor-driven behavior have been hypothesized to require separate neuronal circuitry. Contrary to this notion, innate behavior and olfactory learning were recently shown to share circuitry that includes the Drosophila mushroom body (MB). But how a single circuit drives two discrete behaviors remains unknown. This study defines an MB circuit responsible for both olfactory learning and innate odor avoidance and the distinct dDA1 dopamine receptor-dependent signaling pathways that mediate these behaviors. Associative learning and learning-induced MB plasticity require rutabaga-encoded adenylyl cyclase activity in the MB. In contrast, innate odor preferences driven by naive MB neurotransmission are rutabaga independent, requiring the adenylyl cyclase ACXD. Both learning and innate odor preferences converge on PKA and the downstream MBON-γ2α'1. Importantly, the utilization of this shared circuitry for innate behavior only becomes apparent with hunger, indicating that hardwired innate behavior becomes more flexible during states of stress (Noyes, 2023).

These data reveal the shared use of a discrete circuit for both state-dependent odor-driven behavior and experience-dependent odor learning. The shared components include the upstream DA neurons, the MBn-expressed DA receptor dDA1, and the downstream MBON. Odor response processing for state-dependent behavior and odor learning diverge at the level of the dDA1 receptor-activated adenylyl cyclase, with ACXD employed for innate state-dependent odor driven behavior and rut employed for olfactory learning. The unique activation of rut for olfactory learning is explained by the fact that this adenylyl cyclase functions as a coincidence detector, synergistically responding to both DA receptor activation from the unconditioned stimulus and Ca2+ increases due to the conditioned stimulus (Noyes, 2023).

ACXD is a transmembrane AC that is expressed in a number of tissues including the brain ( and is orthologous to the mammalian AC2. Mammalian AC2 activity is Ca2+ independent. If ACXD is also Ca2+ independent, it would provide a mechanism for the engagement of distinct cAMP pathways by dDA1 for state-dependent versus experience-dependent olfactory behavior. Thus, common neural circuitry is employed for both state-dependent and conditioned behaviors with the unique changes of MBn output influenced by the intracellular signaling pathways that are mobilized. Dopaminergic input to the dDA1 DA receptor expressed in the γ2 compartment of MBn activates an intracellular signaling pathway that includes the ACXD adenylyl cyclase, PKA activity, and the release of ACh. The downstream MBON-γ2α'1 responds to the MB ACh release through the α2 nACh receptor, with the activity of the MBON-γ2α'1 ultimately dictating the balance in state-dependent odor approach/avoidance. The simplest model to account for the state-dependent MBON activity would have the internal state modulating DA input into to the MBn to increase or decrease ACh release onto the MBON. However, the current data failed to detect a significant change in ACh release between the fed and starved conditions. Nevertheless, the activity of the PPL1-γ2α'1 that influences the MB γ2 compartment is required for state-dependent behavioral responses to odor. A proposed model for reconciling these observations envisions that the basal activity of this circuit is required for behavioral-state odor choice but that starvation mobilizes a qualitative or quantitative signal independent of the magnitude ACh release by the MBn to increase MBON activity. An unidentified signal representing hunger could directly enhance MBON excitability. For example, octopamine has been proposed as a feeding signal that acts directly on MBONs (Noyes, 2023).

Similarly, a hunger signal could act on neurons elsewhere in the brain that ultimately connect to MBONs through intermediary neurons. The hunger-responsive neuropeptide leucokinin acts on DAns that connect to MBONs (Noyes, 2023).

Alternatively, there may be a co-neurotransmitter released by the MBn due to starvation that works to increase MBON activity. Finally, the possibility that starvation does modulate MBn ACh output is left open, since the reporters employed lack the sensitivity to detect this change. Future investigations into state-dependent changes in MBON-γ2α'1 physiology will need to grapple with numerous competing hypotheses (Noyes, 2023).

Changes in odor responses in MBONs have suggested that learning induces a change in connectivity in the MBn-MBON synapse. In addition, compartment specific plasticity in MB ACh release was discovered that fits with the idea that plasticity observed in MBONs occurs from the input of MB compartments (Noyes, 2023).

However, there has been a lack of data connecting the MBn ACh release plasticity with MBON plasticity and particularly to the central role for the rut adenylyl cyclase. The current data offer this important connection. It shows that the MB γ2 compartment undergoes a rapid depression in response to odor/shock pairings during aversive learning and that rut is required for the acquisition of this depression. Downstream of the MB γ2 compartment, MBON-γ2α'1 drives approach and also undergoes a learning-induced depression (Noyes, 2023).

These results put prior speculation about how the genetic regulation of cAMP signaling through the rut adenylyl cyclase drives Drosophila memory on concrete ground. The work does not conclusively delineate a role for dDA1 in the MB plasticity. Loss of MB dDA1 dramatically reduced naive odor responses in MB γ2. This precluded attempts to measure dDA1 effects on MB γ2 depression because the naive responses were already low. Interestingly, both learning- and starvation-dependent odor avoidance require PKA. A likely explanation is that rut and ACXD are spatially segregated, creating distinct cAMP microdomains or signaling platforms. Thus, PKA activity would result in the phosphorylation of unique substrates within those microdomains (Noyes, 2023).

The characterization of the MB as a brain region for learned, but not innate, olfactory behavior was motivated by experiments eliminating MBn or blocking MB output. Disrupting the MB eliminates odor-associated memory but has no effect on innate avoidance of those same odors. Recent work has overturned this simple categorization demonstrating some DANs, MBONs, and MBns do contribute to innate olfactory behavior in certain circumstances. Interestingly, the majority of reports define a role for MBns in innate behavioral responses to food-related odors (Noyes, 2023).

The results, using more general, non-food odors, puts the hypothesis that MBn regulates innate behavior on more solid ground. Importantly, it was found that MB dDA1 is required for state-dependent behavior to general odors. This is in contrast to a report finding that dDA1 is not involved but that DAMB is required for state-dependent behavior to food odors (Noyes, 2023).

This difference will be a key element to understand state-dependent behavior moving forward. The Drosophila and mammalian olfactory systems are remarkably similar in terms of anatomical organization and function. In both, odorant molecules activate olfactory sensory neurons (OSNs), with each OSN only expressing one type of odorant receptor (OR). Each OSN expressing the same type of OR project to the same glomeruli (Noyes, 2023).

Within the glomeruli, the OSNs synapse onto projection neuron (PN) dendrites, and PN activity is modified in the glomeruli by local inhibitory interneurons before being sent on to multiple higher-order brain regions. PN neurons connect to downstream neurons in the mammalian piriform cortex and in the Drosophila MB in a seemingly random manner. Like the Drosophila MB, the piriform cortex is critical for olfactory memory. It is not clear how the piriform cortex is involved in state-dependent olfactory behavior. However, in humans, odor coding changes in the piriform cortex with hunger and sleep deprivation, and piriform cortex neuron activity levels are inversely correlated with sexual satiety in rats (Noyes, 2023).

It is concluded from these results demonstrating dDA1-dependent MBn Ach release and a dDA1-dependent MBON-γ2α'1 Ca2+ in response to odor that dDA1 directly modulates the MBn/MBON connection. However, due to a limitation in the sensitivity of the ACh sensor employed, it was not possible to directly record ACh input to MBON-γ2α'1. Based on the established direct cholinergic connection between these MBn and MBONs and the lack of any known non-MB cholinergic innervation to this brain region, it is believed that the conclusions are merited. However, a formal possibility that other intermediary neurons mediate this relationship must be left open (Noyes, 2023).



This study describes the structural and functional properties of the promoter region of a dopamine receptor-gene (Dmdop1) from Drosophila. The transcriptional start site was identified by 5'-RACE (5'-rapid amplification of cDNA ends) cloning and primer-extension analysis. A consensus site for transcriptional initiation (INR element) is located 494 bp upstream of the ATG codon of the open reading-frame. The promoter neither contains TATA- nor CAAT boxes but several GC-rich elements. Relative promoter activity was monitored by CAT reporter-gene analysis in different neuronal cell lines. The Dmdop1 promoter contains one activating (-454/+125) and two silencing regions (-1481/-454 and +125/+495). Interestingly, one silencing region harbours a CRE (cAMP responsive element) site. Since the DmDOP1 receptor leads to cAMP production in cells, the CRE site might contribute to the receptors' own expression by cAMP-dependent transcription factors (Kehren, 2005).

Physiological functions of Dopamine in Drosophila

Requirement of circadian genes for cocaine sensitization in Drosophila

The circadian clock consists of a feedback loop in which clock genes are rhythmically expressed, giving rise to cycling levels of RNA and proteins. Four of the five circadian genes identified to date influence responsiveness to freebase cocaine in the fruit fly, Drosophila melanogaster. Sensitization to repeated cocaine exposures, a phenomenon also seen in humans and animal models and associated with enhanced drug craving, is eliminated in flies mutant for period, clock, cycle, and doubletime, but not in flies lacking the gene timeless. Flies that do not sensitize owing to lack of these genes do not show the induction of tyrosine decarboxylase normally seen after cocaine exposure. These findings indicate unexpected roles for these genes in regulating cocaine sensitization and indicate that they function as regulators of tyrosine decarboxylase (Andretic, 1999).

In response to exposure to volatilized freebase cocaine, Drosophila perform a set of reflexive behaviors similar to those observed in vertebrates, including grooming, proboscis extension, and unusual circling locomotor behaviors. Additionally, flies can show sensitization after even a single exposure to cocaine provided that the doses are separated by an interval of 6 to 24 hours. Sensitization, a process in which repeated exposure to low doses of a drug leads to increased severity of responses, has been linked to the addictive process in humans and is potentially involved in the enhanced craving and psychoses that occur after repeated psychostimulant administration. Circadian variation occurs in the agonist responsiveness of Drosophila nerve cord dopamine receptors functionally coupled to locomotor output. This variation is dependent on the normal functioning of the Drosophila period (per) gene, the founding member of the circadian gene family. Because changes in postsynaptic dopamine receptor responsiveness are also seen during cocaine sensitization in vertebrates, flies mutant in circadian functions were examined for alterations in responsiveness to cocaine. Wild-type (WT) flies as well as flies containing a per null mutation, pero, were exposed to 75 microg of cocaine four times over 2 days, and the fraction of flies showing severe responses was quantified after each exposure. Whereas WT flies show sensitization after the initial cocaine exposure, pero flies show no sensitization either to a normal or increased dose even after repeated exposures. As with WT flies, pero flies showed a dose-dependent increase in the severity of responses, and the normal cocaine-induced types of behaviors were observed. In other words, pero flies respond to cocaine but do not sensitize (Andretic, 1999).

per alleles that either shorten or lengthen the circadian periods show distinct patterns of cocaine responsiveness. The short-period mutants perS and perT both show increased responsiveness to the initial cocaine exposure and weak sensitization to a second 75-µg exposure, with only the sensitization of perS showing statistical significance. Sensitization is not observed in these lines when tested with other cocaine doses. The long-period mutant perL1 showes a normal initial cocaine response but no sensitization to a subsequent exposure (Andretic, 1999).

Similarly, other circadian genes appear to have effects on cocaine sensitization: Both clock and cycle mutants fail to sensitize when given two doses of cocaine. Because these mutants showed an increased sensitivity to the first exposure, cocaine doses were decreased to 50 µg. The inability of clock and cycle to sensitize is markedly similar to the behavior of pero mutants. The gene product of timeless (tim), Tim, is required for the nuclear translocation of Per and its stability in the cytoplasm; in timo mutants, cytoplasmic Per is degraded and per mRNA levels are constant (Andretic, 1999).

Recently, a doubletime (dbt) protein with homology to human casein kinase Iepsilon was identified and shown to be required for phosphorylation of Per. Cocaine responses were tested in two viable dbt mutants, dbtS and dbtL, which shorten and lengthen the circadian locomotor period, respectively. dbt mutants require a substantially higher cocaine dose to show behaviors normally observed at 75 µg, but even at these higher doses dbt flies do not show significant sensitization. If the role of dbt in cocaine responsiveness is analogous to its role in circadian behavior, then Per phosphorylation status may be important in regulating both initial cocaine responsiveness and sensitization (Andretic, 1999).

Modulation of dopamine receptor responsiveness is important in both the sensitization to cocaine in vertebrates and in the circadian modulation of locomotion in Drosophila. To see whether cocaine-sensitized flies would show an increase in the responsiveness of the nerve cord dopamine D2-like receptors, a test was performed using a preparation of behaviorally active decapitated flies that allows direct addition of drugs to the nerve cord. After decapitation, cocaine-sensitized WT flies locomote significantly more than sham-treated controls in response to the dopamine D2-like agonist quinpirole. However, there was no increase in quinpirole responsiveness of pero flies that did not sensitize to repeated cocaine exposures. Thus, similar to the inability of the pero mutant to modulate receptor responsiveness as a function of the time of day, pero is unable to modulate dopamine receptor responsiveness after cocaine exposure. The observation that cocaine sensitization is associated with increased responsiveness of postsynaptic dopamine receptors shows additional similarities between this system and that in higher vertebrates, where a similar relation holds (Andretic, 1999).

In Drosophila, sensitization requires the trace amine tyramine because the mutant inactive, which is defective in sensitization, shows both reduced tyramine and reduced levels of the enzyme involved in tyramine synthesis, tyrosine decarboxylase (TDC). An active role for tyramine in sensitization is indicated because TDC enzyme activity is induced after a single cocaine exposure, with a time course consistent with that for the development of sensitization. To test if the correlation between induction of TDC activity and behavioral sensitization holds for the circadian mutants, TDC activity was measured in the circadian mutant flies after a single exposure of cocaine. In contrast to WT flies, in which TDC activity is induced after cocaine exposure, the pero, cycle, and clock lines that are defective in sensitization show no such induction; only timo, which shows normal sensitization, induces TDC activity. It thus seems likely that the transcriptional regulator Per, presumably in conjunction with Clock and Cycle, is a direct or indirect regulator of TDC after exposure to cocaine (Andretic, 1999).

The sensitization defects in inactive and per mutant flies can be distinguished by differences in the ability of tyramine feeding to restore sensitization. The locomotor and cocaine sensitization defects in inactive mutant flies can be rescued by feeding tyramine to adults, but the sensitization defect in pero flies is distinct, because feeding tyramine to pero adults does not rescue sensitization. It is presumed that tyramine from the food can enter tyramine nerve terminals in inactive flies, where it is still subject to a cocaine-stimulated release mechanism that mediates sensitization. The failure of tyramine feeding to rescue sensitization in pero flies is most readily understood if the per gene product is required for this regulated release (Andretic, 1999).

Similar to inactive , tyramine increases initial cocaine responsiveness in pero flies. Exposure of tyramine-fed pero flies to 35 µg of cocaine induces behaviors normally seen in control flies exposed to 75 µg. Thus, although long-term increase of tyramine levels can affect initial cocaine responsiveness, it is not sufficient for sensitization in flies lacking normal per function (Andretic, 1999).

A unifying feature of most genes that regulate circadian rhythmicity in Drosophila and vertebrates is the PAS dimerization domain, common to a subset of basic helix-loop-helix transcription factors. Within the circadian cycle, Clock/Cycle heterodimers activate per transcription, whereas Per/Tim heterodimers inhibit the activity of Clock/Cycle. Mutations in per, clock, and cycle share the same cocaine phenotype: a deficiency in the ability to sensitize after one or more drug exposures. This similarity leads to the suspicion that as in circadian behaviors, these genes are functioning in a common pathway. In contrast to the above mentioned genes, the timo mutant showed normal cocaine responses. The implication of this finding is twofold. (1) There must be an as yet unidentified Per binding partner that is specifically involved in regulation of drug responsiveness, and (2) drug responsiveness is likely regulated by per expression in a set of cells distinct from those involved in circadian function. In timo mutants, Per levels are constitutively low; if the same Tim-containing cells were involved in circadian and cocaine responses, timo flies should not sensitize (Andretic, 1999).

Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila

Drugs of abuse have a common property in mammals, which is their ability to facilitate the release of the neurotransmitter and neuromodulator dopamine in specific brain regions involved in reward and motivation. This increase in synaptic dopamine levels is believed to act as a positive reinforcer and to mediate some of the acute responses to drugs. The mechanisms by which dopamine regulates acute drug responses and addiction remain unknown. Evidence that dopamine plays a role in the responses of Drosophila to cocaine, nicotine or ethanol. A startle-induced negative geotaxis assay and a locomotor tracking system were used to measure the effect of psychostimulants on fly behavior. Using these assays, it was shown that acute responses to cocaine and nicotine are blunted by pharmacologically induced reductions in dopamine levels. Cocaine and nicotine showed a high degree of synergy in their effects, which is consistent with an action through convergent pathways. In addition, dopamine was found to beinvolved in the acute locomotor-activating effect, but not the sedating effect, of ethanol. This study shows that in Drosophila, as in mammals, dopaminergic pathways play a role in modulating specific behavioral responses to cocaine, nicotine or ethanol. It is therefore suggested that Drosophila can be used as a genetically tractable model system in which to study the mechanisms underlying behavioral responses to multiple drugs of abuse (Bainton, 2000).

Circadian modulation of dopamine receptor responsiveness in Drosophila melanogaster

The circadian function of Drosophila dopamine receptors was investigated by using a behaviorally active decapitated preparation that allows for direct application of drugs to the nerve cord. Quinpirole, a D2-like dopamine receptor agonist, induces reflexive locomotion in decapitated flies. The amount of locomotion induced changes as a function of the time of day, with the highest responsiveness to quinpirole during the subjective night. Furthermore, dopamine receptor responsiveness is under circadian control and depends on the normal function of the period gene. The head pacemaker is at least partly dispensable for the circadian modulation of quinpirole-induced locomotion, because changes in agonist responsiveness persist in decapitated flies that are aged for 12 h. This finding suggests a role for the period-dependent molecular oscillators in the body in the modulation of amine receptor responsiveness (Andretic, 2000).

The circadian variation in locomotor output of the Drosophila nerve cord in response to dopamine agonist stimulation shows two interesting differences from the pattern of locomotor responsiveness in living flies: (1) the rhythms of quinpirole-stimulated nerve cord responsiveness are in opposite phase with in vivo locomotor activity patterns, reaching peak levels during the subjective night, at the time when living flies are least active; (2) there are subtle differences in the activity profiles of the intact and decapitated flies during the light-to-dark transitions (Andretic, 2000).

The out-of-phase nature of in vivo vs. nerve cord locomotion is most readily explained if the nerve cord responses are modulated as compensatory postsynaptic effects, as has been observed in vertebrates and Drosophila. Postsynaptic dopamine receptors can compensate for differences in the amount of presynaptic release, decreasing sensitivity when dopamine release is high, and increasing when it is low. It has been shown that constitutive overexpression of a stimulatory G-alpha subunit in the dopamine and serotonin neurons, which is expected to increase amine release, results in a decrease in postsynaptic responsiveness to quinpirole. Reciprocally, overexpression of the inhibitory G-alpha subunit or tetanus toxin results in an increased responsiveness to quinpirole. It is speculated that increased dopamine release during the subjective day would stimulate locomotor behaviors, with decreased postsynaptic receptor sensitivity acting as a partially effective compensatory mechanism. At night when dopamine release is presumed to be lower, up-regulation of receptor responsiveness would mediate the enhanced response to quinpirole. Although this model postulates that dopamine release is under circadian control, dopamine synthesis is not, because no variation in brain dopamine content as a function of the 24-h day is found. If modulated dopamine release sets the responsiveness of the quinpirole-sensitive receptors, it could occur even in flies lacking brain input. In invertebrates, the ventral cord, unlike the higher vertebrate spinal cord, contains aminergic cell bodies. Thus, a rhythm of aminergic release under the control of body modulatory circuits could set the responsiveness of quinpirole-sensitive receptors. In intact flies, circadian behavior could be under coordinated control of both the body oscillators and the pacemaker in the brain, because additional dopamine input to the ventral cord originates in the brain and reaches the nerve cord through descending dopamine fibers. Alternatively, the observed circadian modulation of quinpirole sensitivity could be under more direct circadian control. By this scenario, modulation of dopamine receptor sensitivity or other signaling components downstream of the receptor would be modulated, independent of the magnitude of dopamine release (Andretic, 2000).

These results are most simply consistent with a role for quinpirole-activated dopamine receptors acting in the output pathway from the brain circadian pacemaker. However, none of the results preclude a role for these as of yet unidentified receptors in modulating intercellular responses between cells of the brain circadian pacemaker. Biogenic amines have been implicated in the control of motor behaviors in vertebrates and invertebrates, both in the central and peripheral nervous system. In humans, the importance of dopamine in motor control is most evident in Parkinson's disease, where degeneration of dopamine cell bodies in substantia nigra results in movement disorders. Interestingly, some Parkinson's disease patients display variations in circadian activity patterns, whereas other studies show daily oscillations in the severity of the symptoms, indicating potential communication between the dopamine system and circadian clock. In spinal cats, where the neural axis has been transected, monoaminergic systems are involved in initiation and modulation of locomotion. In arthropod species, injections of dopamine, serotonin, or octopamine into the central nervous system evokes distinct motor postures, suggesting that they are released endogenously to mediate behavior (Andretic, 2000 and references therein).

Data indicate a role for modulation of dopamine receptor responsiveness in circadian behavior. Modulation of dopamine receptor sensitivity is involved in modulating responses to the indirect amine agonist cocaine, both in vertebrates and in flies. Cocaine functions as a stimulator of reflexive motor and locomotor behaviors both in flies and in vertebrates. It is thus not totally surprising that modulation of responsiveness to cocaine in Drosophila crucially depends on the normal function of a subset of the circadian genes. Given this overlap in functions, it seems likely that there will be altered circadian functions in other mutants showing altered cocaine responses (Andretic, 2000 and references therein).

Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase

Dopamine (DA) is the only catecholaminergic neurotransmitter in Drosophila. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH or pale) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. ple rescue experiments were performed with modified DTH transgenes. The results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, a GAL4 driver transgene, TH-GAL4, was generated containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, it was observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. It is proposed that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila (Friggi-Grelin, 2003).

Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila

The catecholamines play a major role in the regulation of behavior. This study investigated the role of dopamine and octopamine (the presumed arthropod homolog of norepinephrine) during the formation of appetitive and aversive olfactory memories. For the formation of both types of memories, cAMP signaling is necessary and sufficient within the same subpopulation of mushroom-body intrinsic neurons. In contrast, memory formation can be distinguished by the requirement for different catecholamines, dopamine for aversive and octopamine for appetitive conditioning. These results suggest that in associative conditioning, different memories are formed of the same odor under different circumstances, and that they are linked to the respective motivational systems by their specific modulatory pathways (Schwaerzel, 2003; full text of article).

These results support three major conclusions: (1) during the association of an olfactory cue with either a sugar reward or an electric shock punishment, both forms of olfactory memories require cAMP signaling within the same 700 Kenyon cells of the MBs; (2) for memory retrieval but not acquisition with either of the two reinforcers, output from this same set of cells is required. Hence, the memory must be formed and stored upstream of this synaptic level. (3) Sugar and electric shock reinforcement are mediated by different modulatory neurotransmitters, DA in case of electric shock and octopamine (OA) in case of sugar reward. These findings confirm and extend previous work, concluding that output synapses of Kenyon cells are the site of olfactory memory. Appetitive and aversive olfactory memories are localized to the same neuropil Associative behavioral adaptations are mediated by the plasticity of synapses within neural circuits. But what are the smallest units of memory? Do they correspond to the modulation of a single synapse or to the concerted change of many or all synapses in a circuit? Attempts to localize olfactory memory in the Drosophila brain have provided partial answers to these questions (Schwaerzel, 2003).

In many species, including Aplysia, mouse, and Drosophila, the type-1 AC has been shown to be critical in synaptic plasticity. No cases of cAMP-independent associative synaptic or behavioral plasticity have yet been reported conclusively. In Drosophila, one of the corresponding mutants, rut, shows abnormal performance in every learning paradigm tested so far. By identifying the minimally sufficient set of neurons that in a rut mutant brain need to express a wild-type form of the RUT protein to restore a particular memory performance, one can localize the memory trace of the corresponding behavioral adaptation. This approach was successfully applied to two types of memory in Drosophila, heat box memory and olfactory memory (Schwaerzel, 2003).

Using the same approach in a side-by-side comparison between sugar and electric shock reinforcement, the current results show that wild-type rut-AC expression in 700 Kenyon cells (25%-30% of total) rescues memory performance for both kinds of reinforcement. Thus, aversive and appetitive olfactory memories are both mediated by synaptic plasticity in the same group of cells (Schwaerzel, 2003).

Attributing the rescue to an effect on synaptic plasticity in the adult Kenyon cells disregards the possibility that the genetic manipulation might rescue a developmental function of rut-AC, necessary later in the adult for memory. Several lines of evidence argue for an adult function, but only recently has a new genetic manipulation been designed that definitely rules out a developmental effect. Use of a temperature-sensitive Gal80, a suppressor of Gal4, ensured that wild-type rut-AC was expressed only during adulthood (Schwaerzel, 2003).

Although the current experiments do not specify where in the Kenyon cells cAMP signaling is required, the existing evidence suggests a presynaptic mechanism at Kenyon cell output synapses. At the Drosophila larval neuromuscular junction, cAMP signaling is presynaptically involved in plasticity. For the sensory-motor synapses mediating classical conditioning of the gill withdrawal reflex in Aplysia, it has been firmly established that the cAMP cascade is involved presynaptically. No conclusive example of a postsynaptic contribution of cAMP signaling has been reported (Schwaerzel, 2003).

In the Aplysia synapses, plasticity has a postsynaptic component based on a mechanism resembling the NMDA receptor and long-term potentiation in mammals. In Drosophila olfactory conditioning, a similar postsynaptic contribution is unlikely to play a role during the first 3 hr, because this effect would require neurotransmitter release from the presynapse, which can be blocked during acquisition and memory retention without a deleterious effect on memory, using shits1, a conditional blocker of synaptic transmission (Schwaerzel, 2003).

The tentative presynaptic effect of cAMP signaling locates the synaptic plasticity underlying olfactory memory to the synapses connecting Kenyon cells to MB output neurons. These are found in the MB lobes, including the rostral peduncle and spur. Additional support for cAMP signaling to occur in the lobes rather than calyx is derived from the 'memory' gene amn, which has been shown to be involved in cAMP regulation. The putative AMN neuropeptide is required exclusively in two prominent neurons, the so-called dorsal paired medial neurons, that profusely innervate the MB lobes. Other components of the cAMP pathway such as rut and receptors for DA and OA, all are predominantly expressed in the adult MB lobes (Schwaerzel, 2003).

Associative synaptic plasticity depends on the convergence between impulses from two signals, the CS and the US. Considering the proposed role of rut-AC as a molecular coincidence detector, one can assume that the MB input neurons carrying the US for sugar and electric shock should also connect to the lobes, although their direct anatomical identification is pending (Schwaerzel, 2003).

The current results show that acquisition of an olfactory memory with electric shock is dependent on the dopaminergic system, whereas acquisition with sugar depends on the octopaminergic system (see Tyramine β hydroxylase). OA as neurotransmitter in sugar learning seems to be conserved between Drosophila and the honeybee. The bee VUMmx1 neuron, an unpaired neuron localized in the subesophageal ganglion, appears to be octopaminergic and has been shown to carry some of the reinforcing properties of the US. It innervates the calices, antennal lobes, and lateral protocerebrum but not the MB lobes. Nevertheless, the learning paradigms used [individual conditioning of the proboscis extension reflex in bees vs the population-based conditioned osmotaxis in Drosophila] are different; therefore, it might be too early to compare the sugar memories in the bee and Drosophila with respect to its organization on a circuit level. Unfortunately, the role of the monoamines in aversive conditioning has not been tested in bees (Schwaerzel, 2003).

These findings raise the question of whether the effects of the two catecholamines on electric shock and sugar learning can be generalized to other appetitive and aversive reinforcers and to positive and negative behavioral modulation in general. In the monkey, midbrain dopaminergic neurons have been described that carry the reinforcing properties of a US in appetitive but not aversive conditioning. It will be interesting to see whether a similar dissociation between modulatory systems for appetitive and aversive conditioning, with the contingency between good-bad and monoamines exchanged, also applies to the monkey, and, potentially, to humans (Schwaerzel, 2003).

Separate memory traces for electric shock and sugar conditioning had been suggested previously, because these have different kinetics of consolidation and decay. The distinct effects of the two catecholamines in the reinforcement pathways discovered in this study underline this notion. Surprisingly, however, localization experiments assign the two memories to the same neuropil structure, a subset of 700 Kenyon cells (Schwaerzel, 2003).

Based on the functional anatomy of the olfactory pathway, odors are assumed to be represented in the MBs by specific sets of Kenyon cells. For an odorant to become predictive of a given reinforcing event (e.g., sugar or electric shock), the output synapses of this set of Kenyon cells should be modified to drive an MB output neuron mediating the conditioned response (e.g., approach or avoidance). MB input neurons representing the appropriate reinforcers (e.g., sugar or electric shock) should provide the modulatory input. At present it is still not known whether the identified monoamines, OA and DA, are the modulatory neurotransmitters at the site of synaptic plasticity or act further upstream in the respective US pathway. The former alternative is supported by the observation that the MB lobes are equipped with DA and OA receptors that can couple to AC of the rut type via Gs protein. In addition, the neurons relevant for electric shock learning send TH-Gal4-positive fibers to the MB neuropil at the level of the spur and the vertical lobes (Schwaerzel, 2003).

The respective output neurons are prespecified to announce sugar or electric shock and so are the modulatory neurons. Hence, these form specific pairs (US-CR pairs) that are functionally linked to adapt the CR neuron to one of many odors in the conditioning events. Two schemes can be proposed of how the US-CR pairs and sets of Kenyon cells might be interconnected. The two diagrams differ with respect to the organization of odor representations in the MBs. If the same odors were represented by several sets of Kenyon cells, each set could be connected with just one US-CR pair. In this case, sugar and electric shock memories could be formed in different sets, both specifically responding to the same odorant, but one being modulated by OA, the other by DA. Both these sets would be contained within the set of 700 Kenyon cells of the 247-Gal4 driver line. Alternatively, if each odor is represented by only one set, the Kenyon cells should be responsive to multiple modulatory inputs. In this case, both memories would be formed within the same cells. The synapses of the US-CR pairs with the Kenyon cells should be closely associated, and these synaptic domains would have to be independently modulated by cAMP signaling. Because Drosophila can associate many events (US) with odors, Kenyon cells may accommodate many such US-CR pairs. A requirement for space at the Kenyon cells may then explain the stalk-like structure of MBs. At present it is not possible to distinguish between these two alternatives (Schwaerzel, 2003).

Dopamine is a regulator of arousal in the fruit fly

Sleep and arousal are known to be regulated by both homeostatic and circadian processes, but the underlying molecular mechanisms are not well understood. It has been reported that the Drosophila rest/activity cycle has features in common with the mammalian sleep/wake cycle, and it is expected that use of the fly genetic model will facilitate a molecular understanding of sleep and arousal. This study reports the phenotypic characterization of a Drosophila rest/activity mutant known as fumin (fmn). fmn mutants have abnormally high levels of activity and reduced rest (sleep); genetic mapping, molecular analyses, and phenotypic rescue experiments demonstrate that these phenotypes result from mutation of the Drosophila Dopamine transporter gene. Consistent with the rest phenotype, fmn mutants show enhanced sensitivity to mechanical stimuli and a prolonged arousal once active, indicating a decreased arousal threshold. Strikingly, fmn mutants do not show significant rebound in response to rest deprivation as is typical for wild-type flies, nor do they show decreased life span. These results provide direct evidence that dopaminergic signaling has a critical function in the regulation of insect arousal (Kume, 2005).

Although fmn flies exhibit such rest/activity phenotypes, there is apparently no effect of the mutation on development or longevity. This contrasts markedly with the results observed for mutations of two other genes that have been implicated in the regulation of Drosophila rest: cyc and Shaker (Sh). Mutations in cyc or Sh reduce life span, relative to genetic background controls, although complete life-span curves were not reported for Sh and there appears to be only a small effect on longevity for the Sh102 allele. The different effects of these mutations on longevity may reflect the relatively selective effect of fmn on arousal. Alternatively, the effects of Sh and cyc mutations on life span might reflect requirements for these genes in developmental or physiological processes other than rest. Cyc protein is a broadly expressed basic helix-loop-helix transcription factor, whereas Sh is a voltage-activated potassium channel with a broad localization in the nervous system. In contrast, DAT deficits only affect dopaminergic neuromodulation and therefore might have a less general impact on development and physiological processes (Kume, 2005).

Fmn flies carry a mutation in the Drosophila dopamine transporter gene, indicating that alterations of dopamine signaling are responsible for the observed phenotypes. dDAT functions in the dopaminergic pathway, as shown by (1) dDAT protein has significant sequence similarity to comparable mammalian and invertebrate transporters, (2) dDAT gene expression is restricted to dopaminergic neurons (as expected for a presynaptic transporter), (3) this transporter has a substrate specificity paralleling that of the mammalian DATs, with dopamine and tyramine being the preferred substrates, and (4) the dDAT transporter mediates uptake of dopamine in cell-based assays and responds to dopamine when expressed in Xenopus oocytes (Kume, 2005).

Dopamine is cleared from the synaptic cleft via presynaptic DAT, and DAT mutant mice exhibit altered presynaptic autoreceptor function, dopamine clearance, and biosynthetic rate in addition to behavioral alterations including spontaneous hyperlocomotion and hyperactivity. These phenotypes are presumably caused by the elevated persistence of released dopamine in these mice. Similarly, it seems likely that increased dopamine signaling in fmn is responsible for the observed hyperactivity and shortening of the rest phase, regarded as sleep in Drosophila (Kume, 2005).

Previous studies in Drosophila implicate biogenic amines in the modulation of activity. In larval Drosophila, 5-HT, OA, TA, and DA regulate locomotion or the sensory-motor circuitry on which such behavior depends. In adult flies, evidence suggests that DA and 5-HT function to regulate locomotor activity and flight, respectively. The study by Lima (2005) is particularly informative in that it demonstrates state-dependent effects of dopamine neuronal stimulation in behaving flies. In flies with low basal activity, the response to targeted (and transient) stimulation of dopamine neurons is an increased probability of locomotor bouts, whereas a similar stimulation of flies showing high basal activity leads to an inhibition. These results are most consistent with a biphasic role for modulation of locomotor activity by released dopamine, with the highest levels of dopamine release leading to locomotor inhibition. This is in agreement with studies that have examined the responses of flies to the psycho-stimulant cocaine, an inhibitor of aminergic transporters. Cocaine stimulation of flies results in transient stereotypies and hyperactivity that are strikingly similar to those seen after cocaine exposure to vertebrate animals. However, the most severely affected flies become akinesic, consistent with a biphasic effect of high extracellular dopamine (Kume, 2005).

Based on the decreased arousal threshold and the prolonged responses of fmn flies to mechanical stimulation, it is suggested that this mutant is characterized by an arousal state with enhanced alertness associated with the expected increase in extracellular dopamine. The absence of significant rest rebound in fmn supports this conclusion, along with the finding that activity level during each arousal period is normal. This is the first direct evidence that altered arousal threshold and decreased rebound can result from perturbations of dopaminergic signaling. Previous results have indirectly implicated DA in arousal. Those studies examined animals with lesions of dopaminergic neuronal populations, changes in firing rates of dopaminergic neurons as a function of sleep states and arousal, or the actions of wake-promoting drugs, some of which modulate DAT or DA receptor activity. The analysis of fmn shows directly that a selective lesion of DAT, presumably with accompanying increased DA levels, results in an alteration of arousal threshold (Kume, 2005).

It is of interest that mouse DAT mutants, like fly fmn, have abnormal sleep, although arousal sensitivity has not been explicitly examined. DAT mutant mice show enhanced spontaneous locomotor hyperactivity that is greatly enhanced by the stimulating effects of novel environment. More importantly, mouse DAT mutants have significantly increased wake bout duration and moderately increased activity levels during the latter half of the active (night) phase of the diurnal cycle. The extension of the active phase in these mice mimics the phenotype of fmn flies that have a lengthened active period and shortened rest phase during the diurnal cycle. DAT knock-out mice also exhibit altered responses to wake-promoting drugs such as GBR12909, modafinil, and caffeine. Such mice show increased sensitivity to caffeine and decreased sensitivity to GBR12909 and modafinil, suggesting that the latter two drugs act on DAT to promote wakefulness. Modafinil has wake-promoting properties in Drosophila, and it will be of interest to determine whether modafinil action is altered in fmn as it is in DAT mutant mice. A role for DAT and dopaminergic signaling in regulating wakefulness in flies and mice provides additional evidence for a similarity between mammalian sleep and insect rest (Kume, 2005).

Dopamine is probably also important for the regulation of arousal in humans. Parkinson's disease patients, who have reduced dopamine levels, often complain of sleepiness. When treated with L-3,4-dihydroxyphenylalanine (L-DOPA), which increases dopamine levels, they recover from sleepiness, but with excessive L-DOPA, they exhibit insomnia. Moreover, patients and animals with narcolepsy, which show increased wakefulness and excessive sleepiness, show a compensational increase in brain D2-like receptors, suggesting a reduced level of dopamine (Kume, 2005).

Although fmn mutants have higher basal levels of activity, they nonetheless exhibit diurnal and circadian rhythms in locomotor activity. However, rhythmicity is less evident compared with that observed in wild-type flies, presumably because there is a higher baseline of activity at all times of day and therefore a corresponding decrease in the amplitude of rhythmicity (Kume, 2005).

Part of the evidence that fmn is a mutation in dDAT comes from transgenic rescue using pan-neurally driven expression of dDAT. It is notable that this rescue is only partial. One obvious explanation for this partial rescue is that expression of ELAV-GAL4 in the dopamine neurons, the sole site of dDAT localization, is weak. However, even stronger dopamine neuron expression of dDAT, under the control of TH-GAL4, yields even weaker rescue. This counterintuitive result may indicate that the precise level of dDAT in dopamine neurons is critical for normal dopamine homeostasis. It is possible that homeostatic mechanisms potentially overcompensate for high DAT levels and reduced synaptic DA by hyperactivating postsynaptic receptors, as has been seen after inhibition of dopamine and serotonin neurons. Thus, dDAT may be a component of a precisely regulated dopamine homeostatic mechanism that controls arousal threshold and overall activity levels (Kume, 2005).

This study of a Drosophila DAT mutant indicates another striking parallel between Drosophila and vertebrates with regard to the functions of biogenic amine systems. It demonstrates that the regulated reuptake of dopamine by DAT is important for setting arousal threshold. Equally important, the identification of a mutation in the pharmacologically important dopamine transporter opens new avenues for use of this genetically tractable model in pharmacological and behavioral studies (Kume, 2005).

Lowered insulin signalling ameliorates age-related sleep fragmentation in Drosophila

Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly Drosophila. This study investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing Drosophila. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. These findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans (Metaxakis, 2014).

Sleep syndromes are highly prevalent in elderly humans and, with a continuing increase in life expectancy and a greater proportion of elderly people worldwide, effective treatments with fewer side effects are becoming increasingly needed. Sleep in flies shares striking similarities with sleep in humans, including an age-related reduction in sleep quality. This study used Drosophila to examine age-related sleep pathologies and to suppress these pathologies through genetic and pharmacological perturbation of insulin/IGF and TOR signaling (Metaxakis, 2014).

This study has shown that the highly conserved IIS pathway, with roles in growth and development, metabolism, fecundity, stress resistance, and lifespan, also affects sleep patterns in Drosophila. Reduced IIS increases and consolidates night sleep, while decreasing day sleep and inducing day activity. Interestingly, dilp2-3 double mutant flies as well as flies with neuron or fat-body-specific down-regulation of IIS showed no obvious or only mild sleep phenotypes in a previous study, suggesting that a strong and/or systemic reduction in IIS activity may be necessary to induce the activity and sleep phenotypes. Consistently, dilp2-3 double mutant flies have very mild growth, lifespan, and metabolic phenotypes compared to the dilp2-3,5 triple mutant flies used in this study. Reduced IIS activity resulted in increased sleep consolidation in young flies. Importantly, reduced IIS ameliorated the age-related decline in sleep consolidation seen in wild-type flies, thus showing that it is malleable. Contrary to the increased sleep consolidation with reduced IIS, high calorie diets have been reported to accelerate sleep fragmentation. Furthermore, dietary sugar affects sleep pattern in flies. Taken together, these findings reveal a role of nutrition and metabolism in sleep regulation and age-related sleep decline in flies (Metaxakis, 2014).

In humans, several studies suggest a link between nutrition and sleep. The amino acid tryptophan can promote sleep, possibly by affecting synthesis of the sleep regulators serotonin and melatonin. Also, the carbohydrate/fat content of the diet seemingly affects sleep parameters. However, most of these studies are based on correlational methods and small sample size, and it is not yet clear how diet affects sleep. Interestingly, sleep duration can affect metabolism, risk for obesity and diabetes, and even food preference. These findings associate sleep and metabolism; thus, manipulation of nutrient-sensing pathways, such as IIS and TOR signalling, may affect activity and sleep in humans (Metaxakis, 2014).

The transcription factor FoxO is an important downstream mediator of IIS. In C. elegans all aspects of IIS are dependent on daf-16, the worm ortholog of foxO. In contrast, in Drosophila IIS-mediated lifespan extension is dependent on dfoxo, whereas several phenotypes of reduced IIS are dfoxo-independent. Activity and sleep were unaffected by the loss of dfoxo in wild-type flies. In contrast, under low IIS conditions loss of dfoxo specifically affected daytime behaviour, with night time behaviours unaffected. Reduced IIS therefore affects day and night sleep and activity through distinct mechanisms. It also uncouples the effects of IIS on lifespan and on night sleep consolidation, since dfoxo is essential for extended longevity of flies with reduced IIS. dFOXO has been previously shown to increase neuronal excitability, possibly via transcription of ion channel subunits or other (Metaxakis, 2014).

It is suggested that a possible such regulator could be octopaminergic signalling, known to promote arousal in Drosophila. Octopamine, the arthropod equivalent of noradrenaline, regulates several behavioural/physiological processes, including glycogenolysis and fat metabolism, as well as synaptic and behavioural plasticity. Moreover, octopamine can affect sleep by acting on insulin-producing cells in the fly brain, thus linking IIS and sleep/activity. Indeed, this study found that IIS mutants have increased octopamine levels and, importantly, pharmacological inhibition of octopaminergic signalling reverted the increased day activity of IIS mutants. Noteworthy, mRNA expression of octopamine biosynthetic enzymes was not changed, but tyramine levels were significantly reduced, suggesting that increased translation, reduced degradation, or increased activity of the tyramine-β hydroxylase regulates octopamine levels in IIS mutants. In contrast to day activity, increased lifespan of IIS mutants was not affected by pharmacological inhibition of octopaminergic signalling, thus separating longevity from the day activity phenotype (Metaxakis, 2014).

The effect of reduced IIS on day sleep/activity was mediated through AKH, the equivalent of human glucagon, an antagonist of. In flies, AKH coordinates the response to hunger through mobilizing energy stores and increasing food intake, as well as inducing a starvation-like hyperactivity. Loss of AKH receptor (AkhR) abrogated the increased activity of IIS mutants without affecting night sleep. These results demonstrate that day and night phenotypes of IIS mutants can be uncoupled, suggesting that the increased night sleep of IIS mutants is not just a compensatory consequence of increased day activity (Metaxakis, 2014).

dilp2-3,5 mutants have increased octopamine levels, and loss of AkhR in the dilp2-3,5 mutant background reduced their octopamine level back to wild-type levels, suggesting that AkhR-mediated regulation of octopamine controls day hyperactivity in IIS mutants. In support of these findings, octopaminergic cells mediate the increased activity effect of AKH in other insects. Flies lacking dFOXO did not respond to chemically induced AKH release, suggesting that AKH affects activity through dFOXO. Therefore, it is suggested that dFOXO and AkhR act through overlapping mechanisms to enhance octopaminergic signalling and induce activity (Metaxakis, 2014).

In flies, AkhR is highly expressed in fat body and its loss alters lipid and carbohydrate store levels. Therefore, AkhR might indirectly enhance octopaminergic signalling through alterations in lipid and carbohydrate metabolism. In support of this idea, lipid metabolism affects sleep homeostasis in flies. Additionally, AkhR expression in octopaminergic cells could regulate octopamine synthesis and release in flies. Interestingly, expression of AkhR is altered in dfoxo mutants, thus implicating dFOXO in AkhR regulation. Both are highly expressed in fat body, an important organ for metabolism in flies, and fat-body-specific insulin receptor may regulate AkhR function through dFOXO activation (Metaxakis, 2014).

In larval motor neurons, dFOXO increases neuronal excitability and octopamine increases glutamate release, suggesting there is at least a spatial functional link between the two. Thus, together with a possible role in AkhR synthesis, dFOXO could act downstream of octopamine to increase activity (Metaxakis, 2014).

To determine the mechanism underlying the IIS-dependent amelioration of age-related sleep decline, downstream components and genetic interactors of IIS were investigated. One such interactor that affects health and ageing is TORC1. TORC1 is a major regulator of translation, through S6K, 4E-BP, and of autophagy, through ATG1. Inhibiting TOR signalling, and thus translation, by rapamycin treatment in wild-type flies recapitulated the sleep features of IIS mutants, even in old flies. This rescue of sleep quality was blocked by ubiquitous expression of activated S6K, suggesting that reduced S6K activity is required for the rescue. These findings, together with previous results showing S6K to regulate hunger-driven behaviours, highlight the importance of S6K as a regulator of behaviour in flies. Thus, manipulating TOR signalling can improve sleep quality through S6K (Metaxakis, 2014).

In mammals, rapamycin treatment has beneficial effects on behaviour throughout lifespan. Although complete block of TOR activity is detrimental for long-term memory, a moderate decrease through rapamycin treatment can improve cognitive function, abrogate age-related cognitive deterioration, and reduce anxiety and depression. Moreover, increased TOR activity throughout development is detrimental for neuronal plasticity and memory. In flies, rapamycin prevents dopaminergic neuron loss in mutants with parkinsonism. Although the role of TOR in brain function has not been well studied in flies, the advantageous effect of rapamycin in both mammalian brain function and sleep in flies may be mediated through common neurophysiological mechanisms (Metaxakis, 2014).

Gene expression studies have suggested that protein synthesis is up-regulated during sleep, which may be an essential stage in macromolecular biosynthesis. Consistent with this, inhibiting protein synthesis in specific brain domains prolongs sleep duration in mammals, suggesting that sleep is maintained until specific levels of biosynthesis occur and aids in explaining the ubiquitously conserved need for sleep. Brief cycloheximide treatment has been shown to prolong night sleep and increased consolidation in flies, indicating an evolutionarily conserved role for protein synthesis inhibition on sleep regulation. Contrary to reduced IIS, cycloheximide reduced day activity, possibly due to the global effect of cycloheximide on protein synthesis or due to toxic defects in flies' physiology. Decreased protein synthesis rates may enhance the necessity for increased sleep duration, to allow sufficient synthesis of proteins and other macromolecules during sleep, allowing organisms to be healthy and functional during the day (Metaxakis, 2014).

Alternatively, the effect of protein synthesis inhibition on night sleep could be the result of reduced expression of specific sleep regulators. This study found that DopR1 and dilp2-3,5 mutants share night phenotypes and that rapamycin did not affect sleep of DopR1 mutants, suggesting that TOR acts on dopaminergic signalling to affect night sleep. Reduced IIS elevated expression of DopR1, independently of dFOXO, in accordance with data from mammals. This effect may be feedback caused by down-regulation of dopaminergic signalling in IIS mutants, although not through direct regulation of DopR. Under normal physiological conditions, dopamine signalling is determined by the level of extracellular dopamine and the rate of DAT-mediated dopamine clearance from the synaptic cleft. The rate of dopamine clearance is dependent on the turnover rate of DAT and the number of functional transporters at the plasma membrane. This study found that reduced IIS and rapamycin treatment induced increased expression of DAT, suggesting an increased rate of dopamine clearance from the synaptic cleft, and thus a reduction in the amplitude of dopamine signalling, without changes in total dopamine levels. DAT function and IIS have recently been linked in mammals. DAT function increases upon insulin stimulation and is diminished on insulin depletion, through alterations in DAT membrane localization. However, IIS-dependent regulation of DAT subcellular localization in Drosophila has not yet been demonstrated. The current data suggest down-regulating dopaminergic signalling, either by loss of DopR1 or increasing DAT levels, is beneficial for sleep quality. In agreement with this it was shown that artificially increasing dopaminergic signalling, through short-term methamphetamine treatment, increases both day and night activity and reduces night sleep, and reverts the beneficial effect of reduced IIS on night behaviours. In mammals, cocaine administration, which enhances dopaminergic signalling, increases TOR activity. Also, rapamycin blocks cocaine-induced locomotor sensitization. Interestingly, cocaine stimulates S6K phosphorylation in rat brains, and this effect is blocked by rapamycin. Taken together, these results show that in flies and mammals dopaminergic and IIS/TOR signalling may interact in similar ways (Metaxakis, 2014).

In conclusion, reduced IIS extends lifespan in diverse organisms. This study has have shown that it can also ameliorate age-related sleep fragmentation, but that the mechanisms by which it does so are distinct from those by which it extends lifespan. Reduced IIS affected day activity and sleep phenotypes through increased octopaminergic signalling, but enhanced octopaminergic signalling did not increase lifespan. Similarly, in Drosophila increased lifespan from reduced IIS requires dfoxo, but the night sleep phenotypes of IIS mutants were independent of this transcription factor. Reduced IIS thus acts through multiple pathways to ameliorate different aspects of loss of function during ageing. IIS links metabolism and behaviour through its components, such as S6K and dFOXO, which act through different neuronal circuits and neurons to affect sleep. The strong evolutionary conservation of these circuits and their functions suggests that pharmacological manipulation of IIS effectors could be beneficial in treatments of sleep syndromes in humans (Metaxakis, 2014).

Punishment prediction by dopaminergic neurons in Drosophila

The temporal pairing of a neutral stimulus with a reinforcer (reward or punishment) can lead to classical conditioning, a simple form of learning in which the animal assigns a value (positive or negative) to the formerly neutral stimulus. Olfactory classical conditioning in Drosophila is a prime model for the analysis of the molecular and neuronal substrate of this type of learning and memory. Neuronal correlates of associative plasticity have been identified in several regions of the insect brain. In particular, the mushroom bodies have been shown to be necessary for aversive olfactory memory formation. However, little is known about which neurons mediate the reinforcing stimulus. Using functional optical imaging, it has been shown that dopaminergic projections to the mushroom-body lobes are weakly activated by odor stimuli but respond strongly to electric shocks. However, after one of two odors is paired several times with an electric shock, odor-evoked activity is significantly prolonged only for the 'punished' odor. Whereas dopaminergic neurons mediate rewarding reinforcement in mammals, these data suggest a role for aversive reinforcement in Drosophila. However, the dopaminergic neurons' capability of mediating and predicting a reinforcing stimulus appears to be conserved between Drosophila and mammals (Riemensperger, 2005; full text of article).

How do these results fit into the current model of associative olfactory learning in Drosophila? Most data are in favor of a coincidence of CS and US onto the presynapses of Kenyon cells and a resultant strengthening of the synaptic efficacy from Kenyon cells to mushroom-body-extrinsic neurons that ultimately influence the fly's behavior. The data suggest an extension of that model by pointing to an as-yet-anatomically-unidentified excitatory feedback loop from Kenyon-cell output neurons onto DA neurons. As a consequence of this proposed model, during the course of CS-US association an odor stimulus of little relevance gains a relevance that is subsequently represented in a prolonged activation of the DA neurons mediating aversive reinforcement. Of course, the possibility cannot be excluded that other brain regions innervated by DA neurons exhibit similar predictive features. However, these predictive features do not seem to be necessary for the initial formation of an aversive memory because mushroom-body output is needed only for retrieval, not for the formation of an aversive olfactory memory (Riemensperger, 2005).

Interestingly, the phenomenon of predictive features of DA neurons resembles the predictive properties of vertebrate DA neurons. The most obvious difference, however, is the fact that DA neurons mainly mediate rewarding reinforcement in vertebrates, whereas in Drosophila DA release is dispensable for appetitive learning (Schwaerzel, 2003). In contrast, octopamine is involved in appetitive olfactory learning in Drosophila and honeybees. In bees, US mediating and predicting properties in the context of appetitive olfactory conditioning have been observed in an identified octopaminergic neuron. It will be an interesting future experiment to test whether DA neurons in Drosophila do indeed respond exclusively to aversive stimuli or whether they respond to reinforcing stimuli in general (Riemensperger, 2005).

In this context it is conspicuous that calcium activities evoked by an electric shock outlast the stimulus. If the calcium activities revealed by these experiments reflect effective time windows for CS-US coincidence, one would expect that backward pairing of US and CS with short interstimulus intervals should also lead to aversive learning. Indeed, This has indeed shown to be the case. However, when backward pairing is performed with longer US-CS intervals (30 s), an approach behavior toward the backward-conditioned odor can be observed, with the functional implication that the odor may now indicate the absence of the punishment. It will be of interest in the future to test whether such a conditioned approach behavior involves also DA neurons (Riemensperger, 2005).

A second difference with observations in vertebrates concerns the fact that in mammals the responsiveness of DA neurons to the increasingly predicted US diminishes during the course of the training and shifts toward the predicting CS. There is no evidence for such a phenomenon in Drosophila because the responses to the first US presentation and US in the last training trial are indistinguishable in amplitude. In this respect, the reinforcement system in insects might be significantly simpler than in vertebrates. Alternatively, the training procedure used in Drosophila could be too short to detect such a phenomenon, or the US used could be too strong. Despite those differences, these data suggest that a reinforcing and US-predicting role of DA neurons represents a common principle of complex brains rather than a specific feature of the mammalian central nervous system (Riemensperger, 2005).

Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae

During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, the question of which neurons attribute these values to odor stimuli was addressed. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. Transgenically expressed channelrhodopsin-2, a light-activated cation channel, was used as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. Distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning (Schroll, 2006; full text of article).

This study addressed a question of central interest in neuroscience: are there distinct neuronal subsystems mediating opposing types of reinforcement? In vertebrates, the activity of dopaminergic neurons reflects reinforcing properties of rewarding stimuli. Conversely, serotonergic neurons have been proposed to mediate aversive reinforcement, but clear evidence is still lacking. In addition, the sufficiency for any neuronal population's activity to substitute for reinforcing stimuli in vertebrates has not been demonstrated. A more informative basis for opposing reinforcement systems is provided from experiments on diverse insect species. In adult Drosophila, dopaminergic neurons respond to a punishing electric shock stimulus, and blocking synaptic transmission from dopaminergic neurons during olfactory learning impairs aversive but not appetitive memory formation. In accordance with these findings, dopamine receptor antagonists disrupt aversive but not appetitive olfactory learning in crickets. In contrast, octopamine has been shown to be a necessary transmitter for appetitive olfactory learning in adult Drosophila and crickets. The sufficiency of a neuronal cell type for mediating a reinforcing stimulus has been demonstrated for honey bees: electrophysiological stimulation of a single neuron substitutes for an appetitive reinforcing stimulus in olfactory conditioning. This neuron most likely belongs to a cluster of octopaminergic neurons that has been described in honeybees, adult Drosophila, and other insect species. In accordance with these findings, local injection of octopamine also substitutes for the reinforcing stimulus in appetitive olfactory conditioning of honey bees. Therefore, several lines of evidence have led to the idea of two modulatory systems being causative for opposite types of learning in insects. These data provide a direct proof of this concept. It will be of interest to see whether opposing modulatory transmitter systems can be identified in vertebrates as well, a task for which ChR2 might provide a valuable tool (Schroll, 2006).

Cell-type-specific limitation on in vivo serotonin storage following ectopic expression of the Drosophila serotonin transporter, dSERT

The synaptic machinery for neurotransmitter storage is cell-type specific. Although most elements of biosynthesis and transport have been identified, it remains unclear whether additional factors may be required to maintain this specificity. The Drosophila serotonin transporter (dSERT) is normally expressed exclusively in serotonin (5-HT) neurons in the CNS. This study examine the effects of ectopic transcriptional expression of dSERT in the Drosophila larval CNS. A surprising limitation on 5-HT storage was found following ectopic expression of dSERT and green fluorescence protein-tagged dSERT (GFP-dSERT). When dSERT transcription is driven ectopically in the CNS, 5-HT is detectable only in 5-HT, dopamine (DA), and a very limited number of additional neurons. Addition of exogenous 5-HT does not dramatically broaden neuronal storage sites, so this limitation is only partly due to restricted intercellular diffusion of 5-HT. Furthermore, this limitation is not due to gross mislocalization of dSERT, because cells lacking or containing 5-HT show similar levels and subcellular distribution of GFP-dSERT protein, nor is it due to lack of the vesicular transporter, dVMAT. These data suggest that a small number of neurons selectively express factor(s) required for 5-HT storage, and potentially for function of dSERT (Park, 2006).

The simplest explanation for this inability to detect intracellular 5-HT is that there is a cell-type-specific limitation on dSERT function. Arguing for this interpretation is the finding that most if not all neurons will nonspecifically take up 5-HT if incubated at a sufficiently high concentration of 5-HT (10 mM), that lack of expression of the vesicular monoamine transporter dVMAT does not affect the storage ability of neurons, and that no increased levels of the expected 5-HT metabolite N-Ac-5-HT is detected in CNS with ectopic dSERT expression. Part but not all of the limitation is due to limited intracellular diffusion of 5-HT, because incubation in a more moderate concentration of 5-HT, 1 mM, leads to a moderate expansion in the number of cells that can uptake 5-HT, but in a dSERT-dependent manner (Park, 2006).

Nevertheless, this interpretation would stand in contrast to conventional wisdom regarding vertebrate amine transporters. Vertebrate amine transporters can be readily expressed in several types of non-neural transformed cell lines as well as in Xenopus oocytes, with only small differences in their functional properties as a function of cellular host, leading to the general view that the nature of the host cell is not critical. Because dSERT can also be expressed in heterologous cell types including Drosophila S2 cells, it seems that the critical difference between this study versus previous studies is the site of expression, the nerve cord of living flies. There are no analogous amine transporter overexpression studies in the vertebrate nervous system, and it is possible that similar mechanisms might act to limit expression of these transporters to appropriate cell types (Park, 2006).

There is much evidence indicating that amine transmitter transporters are subject to post-translational modifications, including glycosylation and phosphorylation. In particular, roles for protein kinase C (PKC) and phosphatase 2A (PP2A) have become apparent through studies with enzyme activators and inhibitors in SERT-transfected cells, where SERT proteins are rapidly phosphorylated in parallel with transporter internalization and loss of functional uptake capacity. In addition, p38 MAPK activation can stimulate hSERT activity in CHO cells without affecting trafficking to the cell surface (Park, 2006).

Multiprotein complexes that include the transporters, scaffolding proteins, kinases, and phosphatases also can affect their activity and propensity to undergo membrane internalization. Any of these proteins could be candidates for a factor limiting dSERT function to specialized types of neurons in the fly CNS. The only limitation on this factor(s) is that it would not appear to limit the membrane appearance of dSERT, that is, that it does not appear to function at an early step in dSERT Golgi maturation, because GFP-linked dSERT shows similar membrane association at the level of light microscopy in all cell types. In rodents, transient expression of SERT and VMAT2 allows the storage and release of 5-HT in developing thalamocortical projections, despite the absence of serotonin biosynthesis in these cells. It has been assumed that VMAT2 and SERT expression are sufficient to allow uptake of 5-HT synthesized at exogenous sites, and storage in the thalamic neurons. It is possible that the additional 5-HT uptake/accumulation that that was observed following incubation in 1 mM 5-HT in the presumptive adult neuroblasts and their progeny reflects a similar phenomenon, transient expression of dSERT in a subset of developing neurons. More importantly, however, the vast majority of neurons do not accumulate 5-HT even in the presence of exogenous 5-HT. Thus, these data suggest that regulated expression of additional factors may be needed to accomplish a change in neurochemical identity. Cell-type-specific ectopic expression of dSERT in Drosophila will be a favorable tool by which to search genetically for factor(s) limiting 5-HT storage activity (Park, 2006).

Drosophila dopamine synthesis pathway genes regulate tracheal morphogenesis

While studying the developmental functions of the Drosophila dopamine synthesis pathway genes, interesting and unexpected mutant phenotypes were noticed in the developing trachea, a tubule network that has been studied as a model for branching morphogenesis. Specifically, Punch (Pu) and pale (ple) mutants with reduced dopamine synthesis show ectopic/aberrant migration, while Catecholamines up (Catsup) mutants that over-express dopamine show a characteristic loss of migration phenotype. Expression of Punch, Ple, Catsup and dopamine was seen in tracheal cells. The dopamine pathway mutant phenotypes can be reproduced by pharmacological treatments of dopamine and a pathway inhibitor 3-iodotyrosine (3-IT), implicating dopamine as a direct mediator of the regulatory function. Furthermore, these mutants genetically interact with components of the endocytic pathway, namely shibire/dynamin and awd/nm23, that promote endocytosis of the chemotactic signaling receptor Btl/FGFR. Consistent with the genetic results, the surface and total cellular levels of a Btl-GFP fusion protein in the tracheal cells and in cultured S2 cells are reduced upon dopamine treatment, and increased in the presence of 3-IT. Moreover, the transducer of Btl signaling, MAP kinase, is hyper-activated throughout the tracheal tube in the Pu mutant. Finally it was shown that dopamine regulates endocytosis via controlling the dynamin protein level (Hsouna, 2007).

This report demonstrates that genes involved in DA biosynthesis also regulate tracheal cell migration, and that this function is mediated by DA. This is unexpected since DA is normally associated with neuronal function. However, this novel developmental function is not fortuitous because of the strong and highly specific expression of Punch in tracheal cells during the migratory phase of tracheal development. In addition, the ectopic migration tracheal phenotypes in Pu/GTPCH mutants can be rescued by expressing a Pu/GTPCH transgene in developing trachea using a btl promoter, demonstrating the trachea-specific function of the DA pathway. Moreover, the expression of this transgene shows a dosage-dependent progression of phenotypic outcomes. That is, mild expression rescues ectopic migration, while over-expression tips the balance to blocked migration (Hsouna, 2007).

The product of GTPCH enzymatic activity, BH4, is also a stabilizing cofactor of nitric oxide synthase (NOS), which is needed for hypoxia-induced outgrowth of terminal tracheal branches. However, the Pu mutant phenotypes reported in this study are most likely unrelated to this developmental process because of the following reasons: (1) Terminal branching occurs near the end of embryogenesis and during larval development whereas the defects in primary and secondary branch migration occur during mid-embryogenesis. (2) Mutations in ple, which has no role in NOS function, also results in ectopic migration phenotypes. (3) DA and the DA pathway inhibitor can phenocopy the genetic mutants. (4) DA treatment can rescue Pu/GTPCH mutant phenotypes, implicating a direct role of DA in primary and secondary branch migration. Interestingly, DA is enriched in the trachea (Hsouna, 2007).

An inhibitory role for DA in the regulation of cell migration has been reported for a number of cell types over recent years. DA is capable of blocking the migration of vascular smooth muscle cells, a factor in the formation of atherosclerotic lesions, in a process mediated through the D1 class of DA receptors (Yasunari, 2003). DA also was reported to interfere with activated neutrophil transendothelial migration. Similarly, DA, acting through D1 receptors, is reported to reduce the migration of regulatory T cells in damaged neural regions, thereby providing protection against T cell-mediated neurodegeneration (Kipnis, 2004). Finally, DA acting through the D2 class of DA receptors, can inhibit tumor angiogenesis in highly vascularized gastric and ovarian tumors. It would be interesting to determine whether the mammalian GTPCH-TH-DA enzymatic pathways also have a non-neurological function in modulating tubulogenesis during development (Hsouna, 2007).

DA pathway mutants show tracheal abnormalities that are strikingly similar to the perturbations of tracheal cell migration in embryos with abnormalities in FGF signaling. Diminution of DA expression in Drosophila embryos, either by mutations (Pu and ple) or by pharmacological depletion (3-IT treatment), has dramatic effects on the stereotyped migratory behavior of tracheal cells and patterning of the resulting branched structure. Entire branches are misdirected and individual cells move away from the tracheal branches, a phenotype that has been termed 'run-away cells'. Under conditions of excess DA, via mutations (Catsup) or pharmacological modification (DA treatment), the converse phenotype, one of blocked migration, is evident. This phenotype is often associated with clustering of tracheal cells near the stunted ends of tracheal tubes. These opposing phenotypes are correlated with the increased or decreased levels of MAPK activation, the mediator of FGFR signaling (Hsouna, 2007).

The data further demonstrate that DA regulates FGF receptor turnover. Btl;;GFP fusion protein is down-regulated in the presence of DA and up-regulated in the presence of 3-IT, either in trachea or in cultured S2 cells. These results are consistent with the model that DA promotes internalization of Btl/FGFR, leading to its degradation through the endocytic pathway. The role of DA in internalization of FGFR is further suggested by the genetic interaction between the DA synthesis and endocytic pathway genes. Mutations in the human tumor suppressor nucleoside diphosphate kinase (NDK) gene (nm23) are strongly associated with tumor metastatic activity. Its functional homolog in Drosophila, abnormal wing discs (awd), has been shown to genetically interact with a temperature-sensitive allele of the shibire gene (shits), which encodes dynamin, a large GTPase required for the formation of clathrin-coated endocytic vesicles. A function for awd/nm23 in the migratory phase of tracheal development has been demonstrated and functional interactions occur with shi/dynamin that are required for the modulation of FGFR/Btl levels in tracheal cells. This report shows that Pu and ple phenotypes are exacerbated by awd and rescued by btl, while the Catsup phenotypes are rescued by awd but exacerbated by btl. These results indicate that Pu/GTPCH and Ple/TH, and by extension, DA, are positive regulators of endocytosis (Hsouna, 2007).

This analysis was extended by implicating direct involvement of DA in regulating the Shi/dynamin protein itself. Mutations in Pu/GTPCH, which regulates DA pools, result in reduction of the Shi/dynamin levels and, in consequence, the subsequent accumulation of FGFR/Btl in the plasma membranes of tracheal cells, thus accounting for ectopic migratory behavior of these mutant cells. Importantly, down-regulation of Shi/dynamin level in the Pu mutants can be rescued by treatment with DA. A direct correlation between DA and dynamin levels is also demonstrated in cultured S2 cells. Thus, the genetic and pharmacological evidence in this report supports the hypothesis that the diminished DA pools that accompany loss-of-function mutations in the Pu/GTPCH and ple/TH genes result in deficits of DA-mediated signaling necessary for Shi/dynamin accumulation (Hsouna, 2007).

Evidence of a role of DA in receptor endocytosis has emerged recently. For instance, DA can promote VEGFR endocytosis in cultured human endothelial cells. DA D3 receptor-mediated modulation of GABAA receptor and DA-regulated endocytosis of the renal cell Na+, K+-ATPase are similarly dynamin-mediated events. Why is a neurohormone also a mediator of endocytosis in other cell types? It is interesting to consider the possibility that the endocytic activity of DA may in fact be its ancestral function, which was adopted by the neurons and tubular cells later. Indeed, primitive, nerve-less multicellular organisms such as sponge can produce dopamine. The precise mechanism(s) by which DA regulates dynamin assembly is not yet clear. However, DA signaling promotes dynamin stabilization and assembly at the plasma membrane in cultured human kidney cells, and thus endocytosis, by activating protein phosphatase 2A which dephosphorylates dynamin-2. It is possible that a similar mechanism of action occurs in the tracheal cells and future experiments will help to address this possibility (Hsouna, 2007).

Dopamine and octopamine regulate 20-hydroxyecdysone level in vivo in Drosophila

The effects of increased level of dopamine (DA) (feeding flies with DA precursor, L-dihydroxyphenylalanine, L-DOPA) on the level of 20-hydroxyecdysone (20E) and on juvenile hormone (JH) metabolism in young (2-day-old) wild type females (the strain wt) of Drosophila virilis have been studied. Feeding the flies with L-DOPA increased DA content by a factor of 2.5, and led to a considerable increase in 20E level and a decrease of JH degradation (an increase in JH level). The levels of 20E were measured in the young (1-day-old) octopamineless females of the strain Tbetah(nM18) and in wild type females, Canton S, of D. melanogaster. The absence of OA led to a considerable decrease in 20E level (earlier it was shown that in the Tbetah(nM18) females, JH degradation was sharply increased). The effects were studied of JH application on 20E level in 2-day-old wt females of D. virilis; an increase in JH titre results in a steep increase of 20E level. The supposition that biogenic amines act as intermediary between JH and 20E in the control of Drosophila reproduction is discussed (Rauschenbach, 2007).

Dopamine-mushroom body circuit regulates saliency-based decision-making in Drosophila

Drosophila melanogaster can make appropriate choices among alternative flight options on the basis of the relative salience of competing visual cues. This choice behavior consists of early and late phases; the former requires activation of the dopaminergic system and mushroom bodies, whereas the latter is independent of these activities. Immunohistological analysis showed that mushroom bodies are densely innervated by dopaminergic axons. Thus, the circuit from the dopamine system to mushroom bodies is crucial for choice behavior in Drosophila (Zhang, 2007).

Gamma neurons mediate dopaminergic input during aversive olfactory memory formation in Drosophila

Mushroom body (MB)-dependent olfactory learning in Drosophila provides a powerful model to investigate memory mechanisms. MBs integrate olfactory conditioned stimulus (CS) inputs with neuromodulatory reinforcement (unconditioned stimuli, US), which for aversive learning is thought to rely on dopaminergic (DA) signaling to DopR, a D1-like dopamine receptor expressed in MBs. A wealth of evidence suggests the conclusion that parallel and independent signaling occurs downstream of DopR within two MB neuron cell types, with each supporting half of memory performance. For instance, expression of the Rutabaga (Rut) adenylyl cyclase in γ neurons is sufficient to restore normal learning to rut mutants, whereas expression of Neurofibromatosis 1 (NF1) in α/β neurons is sufficient to rescue NF1 mutants. DopR mutations are the only case where memory performance is fully eliminated, consistent with the hypothesis that DopR receives the US inputs for both γ and α/β lobe traces. This study demonstrates, however, that DopR expression in γ neurons is sufficient to fully support short- and long-term memory. It is argued that DA-mediated CS-US association is formed in γ neurons followed by communication between γ and α/β neurons to drive consolidation (Qin, 2012).

Because DopR is thought to mediate the US information, identification of the spatial requirements of this receptor pinpoints the initial site of CS-US coincidence detection. To date, most genetic and circuit manipulations suggest that olfactory memory performance at a given retention interval can be dissected into distinct and independently disruptable mechanisms acting in parallel in distinct neuronal cell types. For example, the STM defects of rut and NF1 can be rescued with expression in γ for rut and β/γ neurons for NF1. Experimental dissections of the circuits required for LTM have suggested a major role for β/γ neurons as well as for ellipsoid body (eb) and DAL neurons. Such findings have been interpreted as supporting the idea of independent signaling for parallel memory traces as well as sequential action in different cell types to support a single memory mechanism. The current findings demonstrate that DopR expression in MBs is sufficient to support both rut-dependent and rut-independent forms of CS-US association leading to STM, as well as to consolidated ARM and LTM. This conclusion also generalizes to three different combinations among five different odors, providing strong evidence that the functional distinctions between KC classes are not artifacts caused by differences in the population of neurons involved in coding each odor percept. With each of these odor combinations and memory phases, there also was no case where expression in α/β or α'/β' populations was sufficient or necessary to provide substantial rescue of dumb2 (a piggyBac insertion in the first intron of the DopR locus) mutants (Qin, 2012).

Together, this set of findings pinpoints the DopR-mediated inputs for STM, MTM, ARM, and LTM to the γ neuron population of MB KCs. This conclusion is consistent with findings from previous attempts to map the subset of DA neurons that convey the US to MBs using either inhibition or activation of neural transmission to block or mimic the US signal. In these studies, the largest magnitude effects were seen with stimulation of MB-MP1, a neuron in the PPL1 cluster of DA neurons (although it should be noted that smaller magnitude effects also were seen for several other DA cell types), which is sufficient to substitute for the US. Although inhibition of MB-MP1 neurons has not been demonstrated to block learning, these DA neurons likely participate in mediating at least a portion of the US stimulus for aversive conditioning. MB-MP1 neurons project to the base of the peduncle, occupied by the axons of α/β neurons and the heel of the MB, which is comprised largely of γ neurons. As an independent validation of the hypothesis that these MB-MP1 neurons provide direct input to γ neurons, the GFP reconstituted across the synapse (GRASP) method was used to visualize putative synaptic connections in the heel between these two cell types (Qin, 2012).

The fact that γ lobe expression of DopR is sufficient to restore not only STM but also both ARM and LTM is noteworthy. Previous attempts to map the neural circuits for olfactory memory have revealed roles for α/β lobes in particular for consolidated memory. Because massed and spaced training experiments consist of repetitive training rather than the single training trial used for STM and MTM, differences in circuit requirements could in principle derive from training paradigm-dependent differences in the CS-US association circuit, as appears to be true for appetitive reinforcement. But this appears not to be the case for DopR function in aversive reinforcement, because full rescue of these consolidated forms of memory were obtained with γ lobe expression of DopR (Qin, 2012).

How can this conclusion be reconciled with the requirement for downstream signaling molecules within α/β lobe neuron, as well as in downstream eb neurons and dorsal-anterior-lateral (DAL) neurons? Three possible explanations are seen, that are not mutually exclusive. First, it is possible that US information is deconstructed into more than one pathway, mediated by different receptors. These could include additional DA receptors, or other neurotransmitter systems such as serotonin. It is worth noting that DA inputs to MBs also have been implicated in hunger/satiety modulation of appetitive memory retrieval, and DopR signaling also has been implicated in several forms of arousal that in principle could represent a component of the reinforcement signal that could be separate from a more specific perceptual representation of the shock experience. The findings nevertheless lead to the conclusion that any additional US information depends critically on DopR-mediated DA signaling in the γ lobe population of neurons. A second possibility worth considering stems from the finding that output from α/β lobe, eb, and DAL neurons are each required for retrieval depending on the retention interval measured. Thus a model cannot be formally ruled out in which all of the functional impacts of various manipulations of α/β lobe derive from defects in retrieval. This would be difficult to fathom for cases such as NF1 rescue of STM and Rut function for LTM, but in principle this interpretation is possible. The third possibility is that consolidation of the γ lobe CS-US association involves signaling within α/β lobe neurons, as well as in downstream eb neurons and DAL neurons. Such a model predicts communication between the γ lobe and the rest of MBs during training and/or afterward (Qin, 2012).

Dopaminergic Modulation of cAMP Drives Nonlinear Plasticity across the Drosophila Mushroom Body Lobes

Activity of dopaminergic neurons is necessary and sufficient to evoke learning-related plasticity in neuronal networks that modulate learning. During olfactory classical conditioning, large subsets of dopaminergic neurons are activated, releasing dopamine across broad sets of postsynaptic neurons. It is unclear how such diffuse dopamine release generates the highly localized patterns of plasticity required for memory formation. This study has mapped spatial patterns of dopaminergic modulation of intracellular signaling and plasticity in Drosophila mushroom body (MB) neurons, combining presynaptic thermogenetic stimulation of dopaminergic neurons with postsynaptic functional imaging in vivo. Stimulation of dopaminergic neurons generated increases in cyclic AMP (cAMP) across multiple spatial regions in the MB. However, odor presentation paired with stimulation of dopaminergic neurons evoked plasticity in Ca2+ responses in discrete spatial patterns. These patterns of plasticity correlated with behavioral requirements for each set of MB neurons in aversive and appetitive conditioning. Finally, broad elevation of cAMP differentially facilitated responses in the gamma lobe, suggesting that it is more sensitive to elevations of cAMP and that it is recruited first into dopamine-dependent memory traces. These data suggest that the spatial pattern of learning-related plasticity is dependent on the postsynaptic neurons' sensitivity to cAMP signaling. This may represent a mechanism through which single-cycle conditioning allocates short-term memory to a specific subset of eligible neurons (gamma neurons) (Boto, 2014).

Dopaminergic neurons are involved in modulating diverse behaviors, including learning, motor control, motivation, arousal, addiction and obesity, and salience-based decision making. In Drosophila, dopaminergic neurons innervate multiple brain regions, including the mushroom body (MB), where they modulate aversive learning, forgetting, state-dependent modulation of appetitive memory retrieval, expression of ethanol-induced reward memory, and temperature-preference behavior (Boto, 2014).

Dopaminergic circuits play a particularly critical role in memory acquisition. During olfactory classical conditioning, where an odor (conditioned stimulus [CS]) is paired with an aversive event (e.g., electric shock; the unconditioned stimulus [US]), dopaminergic neurons respond strongly to the aversive US (Mao, 2009). Dopamine functions in concert with activity-dependent Ca2+ influx to synergistically elevate cyclic AMP (cAMP) (Tomchik, 2009) and PKA (Gervasi, 2010), suggesting that dopamine is one component of a molecular coincidence detector underlying learning. Proper dopamine signaling is necessary for aversive and appetitive memory. Moreover, driving activity of a subset of TH-GAL4+ dopaminergic neurons that differentially innervates the vertical α/α' MB lobes (with less dense innervation of the horizontal β/β'/γ lobes, peduncle, and calyx), is sufficient to induce behavioral aversion to a paired odorant in larvae and adult flies. Conversely, stimulation of a different set of Ddc-GAL4+ dopaminergic neurons, the PAM cluster that innervates mainly the horizontal β/β'/γ lobes, is sufficient to induce behavioral attraction to a paired odorant. Thus, dopaminergic neurons comprise multiple circuits with distinct roles in memory acquisition (Boto, 2014).

Multiple subsets of MB neurons receive CS and US information and express molecules associated with the coincidence detection, making them theoretically eligible to generate dopamine/cAMP-dependent plasticity. Yet only some subsets are required to support memory at any given time following conditioning, leaving open the question of how spatial patterns of plasticity are generated during conditioning. This question has been approached, by using a technique to probe the postsynaptic effects of neuronal pathway activation. Odor presentation was paired with stimulation of presynaptic dopaminergic neurons via ectopic expression of the heat-sensitive channel TRPA1, while monitoring postsynaptic effects with genetically encoded optical reporters for Ca2+, cAMP, and PKA in vivo (Boto, 2014).

The present data demonstrate four major points about how dopaminergic circuits function in neuronal plasticity underlying olfactory classical conditioning. (1) Stimulation of small subsets of dopaminergic neurons evokes consistent, compartmentalized elevations of cAMP across the MB lobes. (2) Broad stimulation of dopaminergic neurons generates broad postsynaptic elevation of cAMP, but Ca2+ response plasticity occurs in discrete spatial regions. (3) Stimulation of TH-GAL4+ neurons and Ddc/R58E02-GAL4+ neurons, which mediate opposing behavioral responses to conditioned stimuli, generates an overlapping pattern of Ca2+ response plasticity in the γ lobe, with additional regions recruited by Ddc/R58E02-GAL4+ stimulation. Finally, (4) the spatial pattern of plasticity coincides with differential sensitivity to cAMP in the γ lobe. Collectively, these data suggest that different subsets of neurons exhibit heterogeneous sensitivity to activation of second messenger signaling cascades, which might shape their responses to neuromodulatory network activity and modulate their propensity for recruitment into memory traces (Boto, 2014).

The data suggest that dopaminergic neurons mediate Ca2+ response plasticity largely in the γ lobe and suggest a potential mechanism for localization of short-term, learning-related plasticity. These data coincide with multiple previous studies that have demonstrated a critical role of γ neurons in short-term memory. Rescue of Rutabaga (Rut) in the γ lobe of rut mutants is sufficient to restore performance in short-term memory, whereas rescue in α/β lobes supports long-term memory. Rescue of the D1-like DopR receptor in the γ lobe is sufficient to rescue both short- and long-term memory in a mutant background, suggesting that the γ neurons mediate the dopaminergic input during conditioning. In addition, stimulating MP1 dopaminergic neurons innervating the heel of the γ lobe is sufficient as an aversive reinforcer. Finally, learning induces plasticity in synaptic vesicle release from MB γ lobes, which depends in part on G(o) signaling (Zhang, 2013). The data support a critical role for the γ lobe in short-term memory. Furthermore, the observation of differential sensitivity of the γ lobe to cAMP might provide an elegant explanation for why it is specifically recruited into short-term memory traces (Boto, 2014).

Direct elevation of cAMP was sufficient to generate localized, concentration-dependent Ca2+ response plasticity in the MB γ lobe in these experiments. Because applying forskolin in the bath is expected to elevate cAMP across the brain, the spatial specificity of the effect is remarkable. This was not an acute effect, because the forskolin was washed out before imaging the first postconditioning odor response. At the concentrations that were tested, only the γ lobe was facilitated. Therefore, it is concluded that the γ lobe is most sensitive to elevation of cAMP, which has the effect of differentially recruiting γ neurons into the representation of short-term memory via dopamine-mediated neuronal plasticity. It is possible that additional signaling cascades are involved in generating learning-related plasticity in α/β and α'/β' neurons, given that no Ca2+ response plasticity was observed in those neurons following forskolin application (Boto, 2014).

The dominant model for cellular mechanisms of olfactory associative learning is that integration of information about the conditioned and unconditioned stimuli are integrated by Rut, which functions as a molecular coincidence detector. This would suggest that MB neurons, which receive CS and US information, would exhibit at least somewhat uniform Ca2+ response plasticity. From this molecular and cellular perspective, the finding that the α/β and α'/β' neurons did not exhibit Ca2+ response plasticity when an odor was paired with stimulation of dopaminergic neurons is surprising. These neurons are theoretically eligible to encode memory, because they receive information about the CS and US. However, the finding that γ neurons differentially exhibit dopamine-dependent plasticity following single-cycle conditioning is consistent with the data from the behavioral experiments. In summary, the present results suggest that differential cAMP sensitivity provides a potential mechanism allowing specific subsets of eligible neurons in an array (γ neurons) to differentially encode CS-US coincidence relative to other subsets (α/β neurons) that also receive CS/US information (Boto, 2014).

Sexually dimorphic recruitment of dopamine neurons into the stress response circuitry

Several previous studies in mammalian systems have shown sexually dimorphic behaviors, neuroendocrine changes, and alterations in neurotransmitter release in response to stress. In addition, men and women are differentially vulnerable to stress-related pathologies, which have led to the hypothesis that the stress response circuitry differs depending on sex. The authors used the genetic tractability of Drosophila to manipulate pre- or postsynaptic dopamine signaling in transgenic animals, which were assayed for several parameters of locomotion and heart rate following exposure to two environmental stressors: starvation and oxidative stress. Their results show significant differences in the stress response for males and females by analyzing heart rate, centering time, and high mobility in addition to other locomotor parameters with translational relevance. These data demonstrate that both pre- and postsynaptic neurons are differentially recruited into the dopaminergic stress response circuitry for males and females. The results also show that the response circuits differ depending on the stressor and behavioral output. Furthermore, a translatable Drosophila model is provided for further elucidation of factors involved in the sexually dimorphic recruitment of neurons into the stress response circuitry (Argue, 2013).

It has been assumed that, because of observed sexually dimorphic differences in susceptibility to stress and stress-related disorders, the stress response circuitry likely differs in males and females. Consistent with this expectation, previous work from this lab has shown that tyrosine hydroxylase levels were altered in response to stress in a manner that was dependent on sex, age, and reproductive status of the population being studied, and that mutant lines carrying specific anatomical defects in the brain responded differently depending upon these same parameters. This study continued an analysis of the sexually dimorphic nature of the dopamine circuitry. These kinds of genetic manipulations, and this type of high throughput, in depth analysis of the behavioral output to stress, are only possible in a genetically tractable organism such as Drosophila. These analyses consisted of three-way ANOVAs to assess the interactions between genotype, sex, and stress. By reducing dopamine levels in select populations of dopamine neurons, it was possible to demonstrate a significant interaction among genotypes, sex, and stress, which supports the conclusion that there are sex differences in the recruitment of dopamine neurons into the stress response circuitry. Similar results were observed by decreasing levels of dopamine receptors, showing that the dopamine signaling in total is sexually dimorphic. The inclusion of two distinct stressors and diverse behavioral outputs permits an extension of the conclusions to show that a sexually dimorphic stress response circuitry is a general phenomenon, not limited to one specific stress, one age group, or a single behavioral output to stress (Argue, 2013).

Starvation and oxidative stress were chosen because, although they are unrelated stressors, both have been linked to the development of specific psychiatric disorders and to have sexually dimorphic effects. Similarly, the behaviors highlighted in this study were chosen because they have been shown to change as a consequence of stress, depression and anxiety in mammals, and have also been shown to be sexually dimorphic. High stress loads can increase risk for, and progression of, cardiovascular disease and can alter parameters of locomotor behavior. In rodents, high emotionality inhibits exploration. In Drosophila and mammals, there is decreased locomotion after adaptation to a novel environment, which is sexually dimorphic. Centrophobism is the tendency to spend more time in closer proximity to the perimeter of the environment rather than in the central open area. In rodents, animals that spend more time in the central zone are considered less fearful and less anxious. In Drosophila this behavior is sexually dimorphic, with females typically displaying more avoidance of the center than males (Argue, 2013).

A recent study in Drosophila suggested that time spent close to the boundaries of the enclosure may be due to a desire to explore the walls rather than an avoidance of the center of the arena; however, in the current experiments only limited vertical movement was observed along the walls. One possible explanation for this could be that in the current experiments the petri dishes used for the open field were painted white, so that the animals could not see outside of their enclosure. Another study also concluded that the preference for the boundaries of the enclosure was not due to centrophobism because the animals also preferred to be in close proximity to the walls when the arena was divided into concentric circles regardless of whether the wall was the outer- or inner-most. However, it must be considered that flies may not have the ability to view their arena on such a global scale and that what is important is the tactile sensation of being close to the wall rather than knowing where they are in terms of the whole arena. Although the specific motivation for this behavior in Drosophila has not yet been determined, it was found that this behavior is altered in response to stress in a sexually dimorphic manner. In addition, as with the other behaviors assayed, there is no qualitative assessment of whether increased time spent in the center zone is indicative of an adapative or maladaptive stress response, anxiety, or fear, only that this behavior is altered in response to stress. Other parameters of locomotion that were observed included duration of time spent stopped, or freezing, which is considered a startle response and is adaptive to prevent detection by a potential predator. Previous reports in mice have suggested that females may have an increased startle response compared to males when threatened by predation. By highlighting specific behaviors that are often used in mammalian models for stress, depression, and anxiety, this analysis establishes Drosophila as a useful model for elucidating the sexually dimorphic effects of stress and affective disorders (Argue, 2013).

The aim of this study was not to determine what constitutes a 'normal' stress response, but rather to focus on demonstrating that the response circuits for stress are different for males and females. Although direct comparisons between the different stressors and behaviors were not shown, differences in the responses clearly indicate that not only are the response circuits sexually dimorphic, but they are also unique for a given stressor and specific behavioral response. The same is also true for the various behavioral parameters. This can be clearly observed by noting differences in the genotypes with significant sexual dimorphisms for each stress and for each behavioral output, and by observing differences in whether the stress resulted in an increase or decrease of a specific response. Although directionality of change was given for each of the specific behaviors, the quality of the responses and any effects of the animals' health and survival were not determined. Although identification of the specific neurons important for the behavioral response for females and males was beyond the scope of this study, this data set provided clusters that would be of particular interest in beginning to specify neurons important for the sexually dimorphic stress response. From the heart rate data, the dopamine neurons targeted by 23y and 201y would be of interest for female heart rate because these lines displayed decreased heart rate in response to oxidative stress, whereas most other lines displayed increases. In addition, the neurons targeted by 103y, although limited, copied the increase in heart rate in response to oxidative stress that was observed for elavC155/THK females. For male heart rate, the neurons targeted by 23y, 103y, 4669, and 854 stood out because these all resulted in increased heart rate following oxidative stress. For time spent in the center zone in response to starvation stress in females, the neurons targeted by 103y and 4669 stood out as having possible importance. Interestingly, these are the same lines that were identified for heart rate in response to oxidative stress, suggesting that these subsets could be involved in the female stress response in general rather than being important for a specific behavior or stressor. It should also be noted that elavC155/THK females displayed an increase in time spent in the center zone that was absent in TH-Gal4/THK females, indicating that the dopamine neurons not targeted by TH-Gal4 could be key for this behavioral response. For the receptor heart rate data, D2R was observed to be important for females, whereas DopR and DopR2 seemed to have greater importance for males. For time spent highly mobile, both DopR and DopR2 appeared to be important for females, whereas knockdown of all three receptors altered male behavior (Argue, 2013).

In addition, greater statistical significance was observed for the presynaptic compared to the postsynaptic studies. This interpretation is somewhat complicated by data suggesting that similar to some mammalian dopamine receptors, the D2R receptor may be located presynaptically and act as an autoreceptor which regulates dopamine release. In spite of this complication, the difference in statistical significance between the two data sets could suggest that postsynaptic dopamine targets are not as sexually dimorphic as presynaptic dopaminergic neurons in the stress response circuitry, or effects with the receptors cancelled each other out because Type I-like and Type II-like receptors have opposing actions (Argue, 2013).



The diverse physiological effects of dopamine are mediated by multiple receptor systems. The dDA1 represents one of the Drosophila dopamine receptors that activate the cAMP cascade. To gain insight into the role of dDA1, a polyclonal antibody was generated against the unique sequence in dDA1 and dDA1 distribution in the central nervous system (CNS) was investigaed. In both larval and adult CNS pronounced dDA1 immunoreactivity is present in the neuropil of the mushroom bodies, a brain structure crucial for learning and memory in insects, and four unpaired neurons in each thoracic segment. In addition, the larval abdominal ganglion contained two dDA1 cells in each segment. This expression pattern appears to be maintained in the condensed adult abdominal ganglion although the precise number and the intensity of staining were somewhat variable. The adult CNS also exhibits intense dDA1 immunoreactivity in the central complex, a structure controlling higher-order motor function, moderate expression in several neurosecretory cells, and weak staining in two unpaired neurons in the mesothoracic neuromere. The dDA1 expression in these areas was detected only in the adult, but not in third instar larval CNS (Kim, 2003).

Ap-let neurons--a peptidergic circuit potentially controlling ecdysial behavior in Drosophila

A set of peptidergic neurons is conserved throughout all developmental stages in the Drosophila central nervous system. A small complement of 28 apterous-expressing cells (Ap-let neurons) in the ventral nerve cord (VNC) of Drosophila larvae co-express numerous gene products. The products include the neuroendocrine-specific bHLH regulator called Dimmed (Dimm), four neuropeptide biosynthetic enzymes (PC2, Fur1, PAL2, and PHM), and a specific dopamine receptor subtype (dDA1). For the PC2, Fur1, and PAL2 enzymes, and for the dDA1 receptor, this neuronal pattern represents the vast majority of their total expression in the VNC. In addition, while Dimm and PHM are present in the peritracheal Inka cells in larvae, pupae, and adults, Ap, PC2, Fur1, PAL2, and dDA1 are not. PC2, PAL2, and DA1 receptor expression are all controlled by both dimm and ap. Previous genetic analysis of animals deficient in PC2 revealed an abnormal larval ecdysis phenotype. Together, these data support the hypothesis that the small cohort of Ap-let interneurons regulates larval ecdysis behavior by secretion of an unidentified amidated peptide(s). This hypothesis further predicts that the production of the Ap-let neuropeptide(s) is dependent on each of four specific enzymes, and that a certain aspect(s) of its production and/or release is regulated by dopamine input (Park, 2004).

One of the Drosophila D1-like dopamine receptors, dDA1 (CG9562), is expressed in a subset of the larval and adult CNS neurons. In the VNC, dDA1 immunoreactivity is evident in a single Dorsal neuron in each thoracic and abdominal hemi-neuromere, and a single lateral neuron in each thoracic hemi-neuromere. This expression pattern is similar to that of the Ap-let group. To test whether dDA1 is expressed in Ap-let neurons, the larval CNSs of apGAL4/UAS-GFP or of c929/UAS-GFP were stained with the dDA1 antibody. All of the dDA1 cells were positive with apGAL4, and with c929, and they included the Dorsal chain and one of the two Tv neurons. This indicates the dDA1-positive T neuron is either Tv or Tvb. When the CNS was double-stained with anti-FMRFa antibody that stains the larval Tv neuron, the dDA1 and dFMRFa immunosignals were in distinct cells. Together, these data indicate that the dDA1-positive cell in the T cluster os the Tvb neuron. Similar to PHM, PC2, Fur1, and PAL2, none of the ap-positive ventral neuron pairs expressed dDA1 immunosignals (Park, 2004).

Specification of neuronal identities by feedforward combinatorial coding

Neuronal specification is often seen as a multistep process: earlier regulators confer broad neuronal identity and are followed by combinatorial codes specifying neuronal properties unique to specific subtypes. However, it is still unclear whether early regulators are re-deployed in subtype-specific combinatorial codes, and whether early patterning events act to restrict the developmental potential of postmitotic cells. This study used the differential peptidergic fate of two lineage-related peptidergic neurons in the Drosophila ventral nerve cord to show how, in a feedforward mechanism, earlier determinants become critical players in later combinatorial codes. Among the progeny of neuroblast 5-6 are two peptidergic neurons: one expresses FMRFamide and the other one expresses Nplp1 and the dopamine receptor DopR. The HLH gene collier functions at three different levels to progressively restrict neuronal identity in the 5-6 lineage. At the final step, collier is the critical combinatorial factor that differentiates two partially overlapping combinatorial codes that define FMRFamide versus Nplp1/DopR identity. Misexpression experiments reveal that both codes can activate neuropeptide gene expression in vast numbers of neurons. Despite their partially overlapping composition, the codes are remarkably specific, with each code activating only the proper neuropeptide gene. These results indicate that a limited number of regulators may constitute a potent combinatorial code that dictates unique neuronal cell fate, and that such codes show a surprising disregard for many global instructive cues (Baumgardt, 2007).

In the developing Drosophila VNC, approximately 90 neurons express the LIM-homeodomain regulator Apterous (Ap), and these represent at least six different cell types. This study focuses on three of the Ap neurons: two cells of the Ap cluster, the Tv cells, which express FMRFa, and the Tvb cells, which together with the dAp cells express DopR. A number of regulators involved in Tv neuron specification have been identified, but to better understand specification of the related Tvb/dAp neurons, it was important to identify the putative neuropeptide gene expressed by Tvb/dAp neurons. The completion of the Drosophila genome led to the prediction of several additional neuropeptide genes, including the Neuropeptide like precursor protein 1-4 genes (Nplp1-4). The validity of these predictions has been confirmed by the identification of expressed sequence tags (ESTs) matching these genes, and by the detection of amidated and secreted peptides in circulation, and/or in brain extracts. Expression of gene products from one of these genes, Nplp1, was found in a set of cells in the VNC reminiscent of the Tvb/dAp neurons. In situ hybridization for Nplp1 verified that these cells indeed correspond to the dAp neurons and to one Ap cluster neuron. To further identify this Ap cluster neuron, antibodies were raised against pro-Nplp1 and against one of the processed and amidated peptides, IPNamide, and a similar pattern was detected. Markers were used for specific subsets of Ap neurons, and the Nplp1-expressing cell in the Ap cluster was identified as the Tvb neuron. In Tvb/dAp neurons, Nplp1 and DopR expression commences in the late embryo (18 h after egg laying [AEL]) and persists at least into the third larval stage. Nplp1 and DopR are thus specifically expressed by the 28 embryonic and larval Tvb/dAp neurons (Baumgardt, 2007).

Recent studies have revealed that ap and dimm are important for DopR expression in Tvb/dAp. It was asked whether these Ap neuron determinants, as well as eya, also affected Nplp1 expression. Nplp1 expression was found to depended on eya, ap, and dimm, and eya also regulates DopR. How is Tv versus Tvb/dAp cell fate then determined? Although both cell types express ap, dimm, and eya, only Tv neurons express dac and have activated the BMP pathway. Could the mere absence of dac and/or BMP activation be sufficient to specify the Tvb/dAp fate? To test this, expression of Nplp1 was analyzed in dac and BMP mutants, but no evidence was found of ectopic Nplp1 expression in Tv neurons. Thus, specification of Tvb/dAp neurons likely requires additional factors restricted to this cell type (Baumgardt, 2007).

The COE family of HLH regulators is highly evolutionary conserved, and is represented in Drosophila by a single member, col (Flybase, knot). COE genes play important roles during nervous system development in Caenorhabditis elegans and vertebrates, and col is expressed in the developing Drosophila central nervous system (CNS), although no function has yet been assigned to it there. The involvement of members of this gene family in nervous system development in other species, and the embryonic CNS expression of col, prompted an investigation of the possible role of col during Ap neuron specification. col has a dynamic expression pattern in the VNC, and focus was initially placed on its expression in mature Ap neurons, at 18 h AEL, and larval stages. At these stages, col is expressed specifically in Tvb/dAp neurons, and expression is maintained in these neurons at least into the third larval stage (see below). This raised the possibility that col plays a role in Tvb/dAp cell fate specification. This notion was supported by the complete loss of Nplp1 and DopR expression in col mutants (Baumgardt, 2007).

Previous studies have addressed the regulatory interactions between several of the Ap neuron determinants. However, these had not been addressed in the case of dac, eya, and dimm. As expected from the late onset of dimm expression, no evidence was found of dimm regulation of Dac or Eya. Similarly, as expected from the mild effect of dac upon FMRFa, dac does not regulate Dimm. In contrast, in eya mutants, a nearly complete loss of Dimm expression was found in the Tv, Tvb, and dAp neurons (Baumgardt, 2007).

With a more complete picture of how previously identified Ap neuron determinants interact genetically, whether col acts upstream, downstream, or in parallel to other Ap neuron determinants was determined. The expression of Col was examined in embryos mutant for these other regulators. In general, no severe effects on Col expression were found. The one exception was in sqz, in which a reproducible increase in the number of Col cells was found. This was, however, expected, since sqz affects the composition of Ap cluster cells, with an increase both in the number of Ap cluster cells (specifically in T1) and an increase in Tvb cells at the expense of Tv cells (in T1-T3). In line with this, an increase was also found in Nplp1 cells in sqz mutants (Baumgardt, 2007).

Does col act upstream of other Ap neuron determinants instead? To address this, the expression of these other regulators was examined in col mutant embryos. This analysis was facilitated by the fact that col2 and col3 mutants, which are both genetically strong alleles, are not protein null, thus allowing for detection of Col in col mutants. In addition, Col is expressed by all four Ap cluster neurons at stage 15. In col mutants, it was found that although sqz and Dac are largely unaffected, ap, Eya, and Dimm are all completely absent from Ap neurons. Because ap, eya, and dimm all regulate FMRFa, the loss of these regulators prompted a look at the expression of FMRFa as well, and as expected, in col mutants, a complete loss was found of FMRFa in the Tv neurons. However, FMRFa is still expressed in the SE2 neurons, a feature common to all identified FMRFa regulators except dimm. These results suggest that Ap cluster neurons are generated in col mutants, but are incompletely specified because they express part of their normal specification code such as dac and sqz, but not other elements of the code such as ap, Eya, Dimm, FMRFa, Nplp1, and DopR (Baumgardt, 2007).

The severe effect of col upon ap, eya, and dimm within all Ap cluster neurons, and upon FMRFa within the Tv neuron, is at odds with the restricted expression of Col in Tvb/dAp neurons at late embryonic stages. Therefore whether Col is more widely expressed at earlier embryonic stages was analyzed, focusing on the Ap cluster neurons. This revealed that although col is restricted to Tvb neurons at stages 17 and 16, expression was observed in all four Ap cluster neurons at stage 15, the stage at which these neurons are first identifiable using ap, Eya, Dac, and sqz as specific markers (Baumgardt, 2007).

Is Col expressed even in the progenitor cells generating Ap cluster neurons? Because ap, Eya, Dac, and sqz are not expressed in Ap cluster neurons prior to stage 15, resolving this issue required the identification of the neuroblast lineage generating the Ap cluster. Extensive work during the last two decades has provided a detailed lineage map of most, if not all, of the 30 neuroblasts found in each hemisegment, and has identified regulatory genes expressed by different neuroblasts. These studies, together with the extreme lateral positioning of early Ap cluster neurons and their appearance only in thoracic hemisegments, allowed use a series of specific markers and determine that the Ap cluster is generated by NB 5-6 -- a lateral-most neuroblast that has been shown to generate larger lineages in the thoracic segments. These results, combined with previous detailed studies of NB 5-6 development, suggested a tentative model for the thoracic NB 5-6 lineage, and lead to placement Col expression within this lineage (Baumgardt, 2007).

Col is expressed by all four newly born Ap cluster neurons and is essential for Ap cluster specification, as evident from the complete loss of ap and Eya expression in col mutants. Col is rapidly down-regulated from three Ap cluster cells and maintained only in Tvb. Is the down-regulation of col important for proper Ap cluster differentiation? To test this, col was misexpressed using the apGAL4 driver, which is not expressed until stage 16, thus maintaining col expression in all four Ap cluster neurons at the time when Col is normally down-regulated. This experiment led to frequent activation of Nplp1 in one additional Ap cluster neuron, and staining for Dimm reveals that this cell is indeed the Tv neuron. FMRFa expression is frequently down-regulated in Tv, but no Tv cells were observed that co-express FMRFa and Nplp1. The finding that col misexpression in the Ap cluster only leads to one ectopic Nplp1 cell and no ectopic Dimm cells indicates that col cannot induce a peptidergic cell fate, at least not with this late driver. However, because the two unaffected cells already are expressing ap and Eya, it was predicted that co-misexpression of dimm, together with col, should trigger Nplp1 expression in all four Ap cluster neurons. This is indeed what was found. In summary, down-regulation of col in three Ap cluster neurons is essential for proper Ap cluster specification (Baumgardt, 2007).

Col is expressed prior to ap and Eya in the Ap cluster neurons, and it is essential for ap and Eya expression within these cells. To address whether col is also sufficient to activate ap and Eya, col was misexpressed in all neurons, using the elav-GAL4 driver. This led to ectopic activation of both ap and Eya. In addition, some activation of Dimm, Nplp1, and DopR expression was found. Although ectopic activation of ap or Eya alone was found in several regions, co-activation was largely confined to neuroblast row 5 -- the anterior region of Gsbn expression. Typically six to ten ap/Eya co-expressing cells were observed in the lateral-most part of row 5. Ectopic activation of ap/Eya, together with Dimm, Nplp1, and DopR, was also confined to lateral-most row 5, i.e., the posterior-most part of gsblacZ cells, and further confined to thoracic segments. Ectopic Nplp1/DopR expression is not overlapping with FMRFa, and there is clear evidence of ectopic Dimm expression, indicating that additional peptidergic neurons are being generated. Ectopic generation of ap/Eya double-expressing cells, i.e., ectopic 'Ap cluster' neurons, was observed already at stage 13, i.e., prior to when Ap cluster neurons are normally born (Baumgardt, 2007).

These results show that col can activate ap and Eya in a number of neurons, but can act to generate bona fide Ap cluster neurons only in a highly context-dependent manner: in lateral, thoracic, row 5 neurons. The appearance of six to ten Eya-expressing cells, but only three to five Nplp1/DopR-expressing cells, and no evidence of ectopic FMRFa expression, suggests that the generation of ectopic Ap cluster neurons is biased toward Tvb (Nplp1/DopR expressing) as opposed to Tv (FMRFa expressing) cell fate. In contrast, although col function depends upon these three positional cues, these results indicate that col is able to override the temporal coding within lateral row 5, and activate Nplp1 and DopR in earlier-born neurons (Baumgardt, 2007).

The loss- and gain-of-function studies place col clearly upstream of ap and eya. Does col act merely to regulate ap and eya in early postmitotic Ap cluster neurons, or does it play additional roles during Ap cluster formation? To further address this issue, attempts were made to 'cross-rescue' col with ap and eya, by expressing ap and eya in a col mutant background. First, as a positive control, attempts were made to rescue col by providing col activity using elav-GAL4/UAS-col. This led to a robust rescue, both of Ap cluster determinants (Eya, ap, and Dimm) and of terminal differentiation genes (Nplp1, DopR, and FMRFa). Similar to the col misexpression experiments, a clear increase was found in 'Ap cluster' neurons, primarily of the Tvb type, as evident from the finding of six to ten ap/Eya- and three to five Nplp1/DopR-expressing neurons per hemisegment. Next, attempts were made to 'cross-rescue' col mutants with ap and eya, and indeed a significant degree of rescue of Ap cluster formation was found, as evident both from Dimm and FMRFa expression. In contrast, no evidence of rescue of Nplp1 or DopR was found in these embryos. Because Col can be detected in the col2 and col3 mutant backgrounds, a Dimm/Col-expressing cell was identified adjacent to the Tv/FMRFa neuron. This indicates that ap/eya can partially rescue Tvb cell fate, but in the absence of col activity, these 'Tvb' neurons do not activate Nplp1. In summary, the finding that in col mutants, ap and eya can partially rescue the Tv cell fate, but not Tvb cell fate, suggests additional roles for col in Tvb specification (Baumgardt, 2007).

The results indicate that Tvb cell fate is not specified by a linear col-->ap/eya-->dimm-->Nplp1/DopR genetic cascade. To further address this issue, the sufficiency was examined of col, ap, and eya to activate Dimm when misexpressed both alone and in combination. These experiments reveal that although col can trigger some ectopic activation of Dimm, there is little effect upon Dimm when misexpressing ap, eya, or ap/eya. In contrast, co-misexpression of col with either ap or eya, and in particular, co-misexpression of all three genes, leads to striking ectopic Dimm expression (Baumgardt, 2007).

Does col play a role even at the final step of Tvb differentiation, i.e., in the activation of Nplp1? Attempts were made to address the possible late role of col by misexpressing it alone and together with other Ap neuron determinants, and then assay its potency in activating Nplp1. Importantly, if there is a simple linear col-->ap/eya-->dimmNplp1/DopR genetic cascade at work, the effect of triple co-misexpression of ap/eya/dimm should not be enhanced by addition of col to this code. However, a striking enhancement was found of ectopic Nplp1 expression when adding col to this code. One particular double co-misexpression combination, col/ap, was more potent than others in activating both Dimm and Nplp1. A likely explanation for this effect is that co-misexpression of col/ap activates significant ectopic eya expression (Baumgardt, 2007).

To further address the late role of col, a transgenic RNA-interference (RNAi) line, (UAS-col-dsRNA), was generated, and attempts were made to suppress col gene activity by crossing this line to apGAL4. Because apGAL4 also drives expression in the developing wing disc, the efficiency was examined of this novel tool in suppressing col gene activity in this tissue. This phenocopied the effects of col mutants on wing development, with a clear L3-L4 wing vein fusion, indicating that this RNAi transgene specifically blocks col gene activity. However, upon analyzing late larval (third instar) CNSs, no effect upon Col expression in Tvb neurons was found, and as expected, no effect was found upon Nplp1 expression. Recent studies reveal that RNAi can be efficiently enhanced by overexpression of components of the RNAi pathway, in particular of the Dicer-2 (Dcr-2) gene. Therefore Dcr-2 was co-expressed with col dsRNA (UAS-Dcr-2/+; apGAL4/UAS-col-dsRNA), and a clear effect was found not only upon Col, but importantly, also upon Nplp1 expression. No obvious effect was found in the first instar larvae, but in third instar larvae, Col expression is specifically and completely lost from all Tvb/dAp cells. This leads to a complete loss of Nplp1 in 44% of Tvb/dAp cells, and strongly reduced expression in the remaining expressing cells. Strikingly, this strong effect upon Nplp1 is not an indirect effect of down-regulation of ap, Eya, or Dimm. As anticipated, col RNAi has no effect upon FMRFa expression in the third instar larvae (Baumgardt, 2007).

Previous studies have identified several regulators acting to specify Tv fate and to control FMRFa expression. Although co-misexpression of parts of this code had been previously tested, all possible combinations had not. Similar to the combinatorial activation of Nplp1 and DopR, it was found that whereas co-misexpression of ap/sqz, ap/dac, or ap/dimm has limited effect upon FMRFa expression, triple co-misexpression of these regulators, and in particular of ap/dimm/dac, leads to a dramatic ectopic activation of FMRFa (Baumgardt, 2007).

The identification of two partly overlapping and highly potent combinatorial codes allowed lead to asking of an important question: Does combinatorial misexpression of these regulators merely lead to a general confusion with a mixed neuronal identity, or are these codes truly instructive and specific? To address this issue, the expression of Nplp1 and FMRFa was examined in the various misexpression backgrounds. Not surprisingly, when common and partial components of these codes are misexpressed, such as ap/dimm (common to both Tv and Tvb/dAp neurons), ectopic activation of both Nplp1 and FMRFa was found in different subsets of cells. However, as a third, and cell-type specific, regulator is added, not only does the amount of ectopic, terminal differentiation gene expression increase dramatically, but less evidence of cross-activation of the inappropriate downstream gene was found. This surprising finding reveals that combinatorial misexpression may act in a highly specific and instructive manner, and that these combinatorial codes may be viewed as potent binary switches for cell fate specification. In addition, for both codes, ectopic activation of FMRFa and Nplp1 is observed in neurons throughout the VNC and brain, and traverses many developmental boundaries, such as anteroposterior, dorsoventral, and mediolateral boundaries (Baumgardt, 2007).

This study has identified a sequential regulatory cascade of combinatorial coding that acts to specify two unique neuronal cell fates during Drosophila CNS development. Combined with previous studies, the findings provide the following model for Ap cluster generation and specification. Neuroblast 5-6 forms in the first wave of neuroblast delamination, at late stage 8, and generates a mixed lineage of glia and neurons. At stage 13, Col expression is turned on specifically in thoracic NB 5-6, in two subsequent ganglion mother cells (GMCs) at stages 13/14, and in the four Ap cluster neurons generated from these GMCs, during stages 14/15. The birth order of the four Ap cluster neurons has not been resolved, and the sibling relationship of Tv and Tvb is thus unclear. When Ap neurons are born, col activates ap and eya, whereas sqz and dac are activated by unknown regulator(s). sqz appears to play an early postmitotic role, apparently acting in the Notch pathway, to ensure proper Ap cluster composition, and sqz mutants display both additional Ap cluster cells (in T1) and additional Tvb cells (in T1-T3). col is rapidly down-regulated from three Ap neurons, but remains expressed in the Tvb, where it acts with ap and eya to activate dimm at stage 16. At late embryogenesis, col acts with ap, eya, and dimm to activate Nplp1 and DopR in Tvb. In the Tv neuron, ap and eya act, apparently independently of col, to activate dimm expression. In the Tv neuron, eya furthermore plays a role in setting up competence to respond to the BMP signal. At stage 17, the Tv axon reaches the dorsal neurohemal organ (DNH) and receives the BMP ligand Gbb that activates the Wit receptor, and then triggers activation of the BMP pathway in the Tv neuron. At 18 h AEL, ap, eya, sqz, dac, dimm, and BMP signaling cooperate to activate FMRFa in the Tv neuron. In addition to their roles in neuropeptide regulation and BMP signaling, both ap and eya act to ensure proper axon pathfinding of Tv neurons (eya), as well as Tvb and dAp neurons (ap and eya). The role that col may play more directly in axon pathfinding has not been resolved due to the fact that the expression of the appropriate axonal markers (apGAL4, Nplp1, DopR, and FMRFa) is completely absent in col mutants. Given the complexity of axon pathfinding, it is anticipated that several other regulators are yet to be identified before a combinatorial code for 'Tv-type' or 'Tvb-type' axonal pathfinding is deciphered. Indeed, no evidence was found that combinatorial misexpression of the abovementioned regulators can dictate axonal projections, because ectopic Nplp1 or FMRFa axons are following many different routes in the VNC. Finally, dimm also plays additional roles to those described above and is necessary for the expression of neuropeptide-processing enzymes in peptidergic neurons. Importantly, dimm acts independently to control expression of the neuroamidase gene PHM in the Tv and Tvb neurons, and dimm is sufficient to activate PHM in most, if not all, VNC neurons. Thus, during the specification and differentiation of the Ap neurons, there exists a remarkable diversity in the division of labor between the identified regulators, with most of them participating in more than one, but never all, of the identified events (Baumgardt, 2007).

This study has identified a multistep process for specifying the Tv and Tvb cell fates. What would be the purpose of this type of sequential combinatorial coding? In other model systems with higher genetic resolution, such as Escherichia coli and yeast, extensive genetic analysis has revealed that this type of sequential gene regulation is quite common, and a recent study in C. elegans suggests it may also function during neuronal specification. These regulatory nodes, also known as feedforward loops (FFL), have been shown to ensure fidelity in gene regulation. For instance, in a simple FFL in which gene A regulates gene B, and A/B then co-operate to regulate C, activation of C depends upon prolonged A expression such that A/B will have time to activate C. If A is only active in a short burst, B may be activated, but C is not, because A/B never co-express for a sufficiently prolonged period of time. The role of col during Ap cluster specification provides an excellent example of a FFL used during neuronal cell fate specification. col is expressed in all four Ap cluster cells and plays an early role in activating ap and Eya, but is only maintained in Tvb, where it plays a later role in activating first dimm, then Nplp1. Maintained expression of col in all four Ap cluster neurons, by driving col expression from apGAL4, leads to activation of the Tvb program also in the Tv neuron. Thus, a burst of col expression has a different informational value than persistent col expression -- general Ap cluster specification versus Tvb specification (Baumgardt, 2007).

Misexpression of each of the two identified combinatorial codes leads to striking ectopic activation of the Nplp1 and FMRFa genes, and two particular aspects of these findings were surprising. First, the global potency of these codes: co-misexpression triggers ectopic FMRFa of Nplp1 in a number of neurons, located in many different anteroposterior, dorsoventral, and mediolateral positions. Thus, it would appear that early regulators mainly act to ensure proper combinatorial coding in each neuron, and play a minor role in restricting cell fate by limiting the cell's competence. Once the proper code is in place, the cell fate specification program will be carried out irrespective of the history of the cell. Second, the striking binary effect of these codes is noteworthy: the change of one single player in a code completely alters target gene choice. For instance, misexpression of ap/dimm/dac leads almost exclusively to strong FMRFa activation, but simply replacing dac with col leads to almost exclusively Nplp1 activation. Thus, it appears that more complete codes not only have great potency, but also have great specificity (Baumgardt, 2007).

Col shows a very dynamic expression pattern in the VNC, exemplified in NB 5-6 by the expression in the neuroblast, in two GMCs, in all four Ap cluster neurons, and finally only in Tvb. This poses three obvious questions: what activates col in the neuroblast, what shuts it down in three of the Ap cluster neurons, and finally, what maintains col in Tvb? As for the activation of col in the late 5-6 neuroblast, row 5 neuroblast determinants, thoracic determinants, and late temporal determinants are obvious candidates. Indeed, current work has identified input from a number of such upstream regulators. It is less clear why col expression is lost from three Ap cluster cells and maintained in Tvb. It is possible that the initial expression in all four Ap cluster cells merely reflects residual expression, as an effect of the activation by earlier determinants acting in the neuroblast. But why is col then maintained in Tvb, and similarly, what maintains eya and ap in all four Ap cluster cells? One simple solution would be autoregulation of each gene. But surprisingly, there is no evidence of autoregulation of col. In addition, no clear evidence was found of cross-regulation between col, ap, or eya, at least not during embryonic stages. Thus, it seems likely that other mechanisms, either unidentified regulators or, perhaps, epigenetic mechanisms, act to ensure the continual expression of these regulators during larval (and perhaps adult) life (Baumgardt, 2007).

Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits

Arousal is fundamental to many behaviors, but whether it is unitary or whether there are different types of behavior-specific arousal has not been clear. In Drosophila, dopamine promotes sleep-wake arousal. However, there is conflicting evidence regarding its influence on environmentally stimulated arousal. This study shows that loss-of-function mutations in the D1 dopamine receptor DopR enhance repetitive startle-induced arousal while decreasing sleep-wake arousal (i.e., increasing sleep). These two types of arousal are also inversely influenced by cocaine, whose effects in each case are opposite to, and abrogated by, the DopR mutation. Selective restoration of DopR function in the central complex rescues the enhanced stimulated arousal but not the increased sleep phenotype of DopR mutants. These data provide evidence for at least two different forms of arousal, which are independently regulated by dopamine in opposite directions, via distinct neural circuits (Lebestky, 2009).

'Arousal', a state characterized by increased activity, sensitivity to sensory stimuli, and certain patterns of brain activity, accompanies many different behaviors, including circadian rhythms, escape, aggression, courtship, and emotional responses in higher vertebrates. A key unanswered question is whether arousal is a unidimensional, generalized state. Biogenic amines, such as dopamine (DA), norepinephrine (NE), serotonin (5-HT), and histamine, as well as cholinergic systems, have all been implicated in arousal in numerous behavioral settings. However, it is not clear whether these different neuromodulators act on a common 'generalized arousal' pathway or rather control distinct arousal pathways or circuits that independently regulate different behaviors. Resolving this issue requires identifying the receptors and circuits on which these neuromodulators act, in different behavioral settings of arousal (Lebestky, 2009).

Most studies of arousal in Drosophila have focused on locomotor activity reflecting sleep-wake transitions, a form of 'endogenously generated' arousal. Several lines of evidence point to a role for DA in enhancing this form of arousal in Drosophila. Drug-feeding experiments, as well as genetic silencing of dopaminergic neurons, have indicated that DA promotes waking during the subjective night phase of the circadian cycle. Similar conclusions were drawn from studying mutations in the Drosophila DA transporter (dDAT). Consistent with these data, overexpression of the vesicular monoamine transporter (dVMAT-A), promoted hyperactivity in this species, as did activation of DA neurons in quiescent flies (Lebestky, 2009).

Evidence regarding the nature of DA effects on 'exogenously generated' or environmentally stimulated arousal, such as that elicited by startle, is less consistent. Classical genetic studies and quantitative trait locus (QTL) analyses have suggested that differences in DA levels may underlie genetic variation in startle-induced locomotor activity (see Carbone, 2006 and Jordan, 2006). Fmn (dDAT; Dopamine transporter) mutants displayed hyperactivity in response to mechanical shocks, implying a positive-acting role for DA in controlling environmentally induced arousal (Kume, 2005). In contrast, other data imply a negative-acting role for DA in controlling stimulated arousal. Mutants in Tyr-1, which exhibit a reduction in dopamine levels, show an increase in stimulated but not spontaneous levels of locomotor activity. Genetic inhibition of tyrosine hydroxylase-expressing neurons caused hyperactivity in response to mechanical startle (Friggi-Grelin, 2003). Finally, transient activation of DA neurons in hyperactive flies inhibited locomotion (Lima, 2005). Whether these differing results reflect differences in behavioral assays, the involvement of different types of DA receptors, or an 'inverted U'-like dosage sensitivity to DA (Birman, 2005), is unclear (Lebestky, 2009).

This investigation has developed a novel behavioral paradigm for environmentally stimulated arousal, using repetitive mechanical startle as a stimulus, and a screen was carried out for mutations that potentiate this response. One such mutation is a hypomorphic allele of the D1 receptor ortholog, DopR. This same mutation caused decreased spontaneous activity during the night phase of the circadian cycle, due to increased rest bout duration. In both assays, cocaine influenced behavior in the opposite direction as the DopR mutation, and the effect of cocaine was abolished in DopR mutant flies, supporting the idea that DA inversely regulates these two forms of arousal. Genetic rescue experiments, using Gal4 drivers with restricted CNS expression, indicate that these independent and opposite influences of DopR are exerted in different neural circuits. These data suggest the existence of different types of arousal states mediated by distinct neural circuits in Drosophila, which can be oppositely regulated by DA acting via the same receptor subtype (Lebestky, 2009).

Previous studies of arousal in Drosophila have focused on sleep-wake transitions, a form of 'endogenous' arousal. This study has introduced and characterized a quantitative behavioral assay for repetitive startle-induced hyperactivity, which displays properties consistent with an environmentally triggered ('exogenous') arousal state. A screen was conducted for mutations affecting this behavior, the phenotype of one such mutation (DopR) was analyzed, and the neural substrates of its action was mapped by cell-specific genetic rescue experiments. The results reveal that DopR independently regulates Repetitive Startle-induced Hyperactivity (ReSH) and sleep in opposite directions by acting on distinct neural substrates. Negative regulation of the ReSH response requires DopR function in the ellipsoid body (EB) of the central complex (CC), while positive regulation of waking reflects a function in other populations of neurons, including PDF-expressing circadian pacemaker cells. Both of these functions, moreover, are independent of the function of DopR in learning and memory, which is required in the mushroom body. These data suggest that ReSH behavior and sleep-wake transitions reflect distinct forms of arousal that are genetically, anatomically, and behaviorally separable. This conclusion is consistent with earlier suggestions, based on classical genetic studies, that spontaneous and environmentally stimulated locomotor activity reflect 'distinct behavioral systems' in Drosophila (Lebestky, 2009).

Several lines of evidence suggest that ReSH behavior represents a form of environmentally stimulated arousal. First, hyperactivity is an evolutionarily conserved expression of increased arousal. Although not all arousal is necessarily expressed as hyperactivity, electrophysiological studies indicate that mechanical startle, the type of stimulus used in this study, evokes increases in 20-30 Hz and 80-90 Hz brain activity, which have been suggested to reflect a neural correlate of arousal in flies (Nitz, 2002; van Swinderen, 2004). Second, ReSH does not immediately dissipate following termination of the stimulus, as would be expected for a simple reflexive stimulus-response behavior, but rather persists for an extended period of time, suggesting that it reflects a change in internal state. Third, this state, like arousal, is scalable: more puffs, or more intense puffs, produce a stronger and/or longer-lasting state of hyperactivity. Fourth, this state exhibits sensitization: even after overt locomotor activity has recovered to prepuff levels, flies remain hypersensitive to a single puff for several minutes. Fifth, this sensitization state generalizes to a startle stimulus of at least one other sensory modality (olfactory). In Aplysia, sensitization of the gill/siphon withdrawal reflex has been likened to behavioral arousal. Taken together, these features strongly suggest that ReSH represents an example of environmentally stimulated ('exogenous') arousal in Drosophila (Lebestky, 2009).

DopR mutant flies exhibited longer rest periods during their subjective night phase, suggesting that DopR normally promotes sleep-wake transitions. These data are consistent with earlier studies indicating that DA promotes arousal by inhibiting sleep (Andretic, 2005, Kume, 2005; Wu, 2008). In contrast, prior evidence regarding the role of DA in startle-induced arousal is conflicting. Some studies have suggested that DA negatively regulates locomotor reactivity to environmental stimuli, consistent with the current observations, while others have suggested that it positively regulates this response. Even within the same study, light-stimulated activation of TH+ neurons produced opposite effects on locomotion, depending on the prestimulus level of locomotor activity (Lima, 2005; Lebestky, 2009 and references therein).

This study has found that DA and DopR negatively regulate environmentally stimulated arousal: the DopR mutation enhanced the ReSH response, while cocaine suppressed it. Furthermore, the effect of cocaine in the ReSH assay was eliminated in the DopR mutant but could be rescued by Gal4-driven DopR expression, confirming that the effect of the drug is mediated by DA. Taken together, these results reconcile apparently conflicting data on the role of DA in 'arousal' in Drosophila by identifying two different forms of arousal -- repetitive startle-induced arousal and sleep-wake arousal -- that are regulated by DA in an inverse manner (Lebestky, 2009).

The finding that DopR negatively regulates one form of environmentally stimulated arousal leaves open the question of whether this is true for all types of exogenous arousing stimuli. The 'sign' of the influence of DA on exogenously generated arousal states may vary depending on the type or strength of the stimulus used, the initial state of the system prior to exposure to the arousing stimulus (Birman, 2005; Lima and Miesenbock, 2005), or the precise neural circuitry that is engaged. Future studies using arousing stimuli of different sensory modalities or associated with different behaviors should shed light on this question (Lebestky, 2009).

Several lines of evidence suggest that endogenous DopR likely acts in the ellipsoid body (EB) of the central complex (CC) to regulate repetitive startle-induced arousal. First, multiple Gal4 lines that drive expression in the EB rescued the ReSH phenotype of DopR mutants. Second, endogenous DopR is expressed in EB neurons, including those in which the rescuing Gal4 drivers are expressed. Third, the domain of DopR expression in the EB overlaps the varicosities of TH+ fibers. In an independent study of dopaminergic inputs required for regulating EtOH-stimulated hyperactivity TH+ neurons were identified that are a likely source of these projections to the EB. Fourth, rescue of the ReSH phenotype is associated with re-expression of DopR in EB neurons. Finally, rescue is observed using conditional DopR expression in adults. Taken together, these data argue that rescue of the ReSH phenotype by the Gal4 lines tested reflects their common expression in the EB and that this is a normal site of DopR action in adult flies (Lebestky, 2009).

A requirement for DopR in the EB in regulating ReSH behavior is consistent with the fact that the CC is involved in the control of walking activity. However, the mushroom body has also been implicated in the control of locomotor behavior, and DopR is strongly expressed in this structure as well. Rescue data argue against the MB and in favor of the CC as a neural substrate for the ReSH phenotype of DopR mutants. Unexpectedly, the nocturnal hypoactivity phenotype of DopR mutants was not rescued by restoration of DopR expression to the CC. Thus, not all locomotor activity phenotypes of the DopR mutant necessarily reflect a function for the gene in the CC (Lebestky, 2009).

Interestingly, Gal4 line c547 expresses in R2/R4m neurons of the EB, while lines 189y and c761 express in R3 neurons, yet both rescued the ReSH phenotype of DopR mutants. Similar results have been obtained in experiments to rescue the deficit in ethanol-induced behavior exhibited by the DopR mutant. Double-labeling experiments suggest that endogenous DopR is expressed in all of these EB neuronal subpopulations. Perhaps the receptor functions in parallel or in series in R4m and R3 neurons, so that restoration of DopR expression in either population can rescue the ReSH phenotype. Whether these DopR-expressing EB subpopulations are synaptically interconnected is an interesting question for future investigation (Lebestky, 2009).

Despite its power as a system for studying neural development, function, and behavior, Drosophila has not been extensively used in affective neuroscience, in part due to uncertainty about whether this insect exhibits emotion-like states or behaviors. Increased arousal is a key component of many emotional or affective behaviors. The data presented in this study indicate that Drosophila can express a persistent arousal state in response to repetitive stress. ReSH behavior exhibits several features that distinguish it from simple, reflexive stimulus-response behaviors: scalability, persistence following stimulus termination, and sensitization. In addition, the observation that mechanical trauma promotes release from Drosophila of an odorant that repels other flies suggests that the arousal state underlying ReSH behavior may have a negative 'affective valence' as well. These considerations, taken together with the fact that ReSH is influenced by genetic and pharmacologic manipulations of DA, a biogenic amine implicated in emotional behavior in humans, support the idea that the ReSH response may represent a primitive 'emotion-like' behavior in Drosophila (Lebestky, 2009).

The phenotype of DopR flies is reminiscent of attention-deficit hyperactivity disorder (ADHD), an affective disorder linked to dopamine, whose symptoms include hyper-reactivity to environmental stimuli. If humans, like flies, have distinct circuits for different forms of arousal, then the current data suggest that ADHD may specifically involve dopaminergic dysfunction in those circuits mediating environmentally stimulated, rather than endogenous (sleep-wake), arousal. Given that DA negatively regulates environmentally stimulated arousal circuits in Drosophila, such a view would be consistent with the fact that treatment with drugs that increase synaptic levels of DA, such as methylphenidate (ritalin), can ameliorate symptoms of ADHD (Lebestky, 2009).

In further support of this suggestion, in mammals, dopamine D1 receptors in the prefrontal cortex (PFC) have been proposed to negatively regulate activity, while D1 receptors in the nucleus accumbens are thought to promote sleep-wake transitions. Numerous studies have linked dopaminergic dysfunction in the PFC to ADHD. While most research has focused on the role of the PFC in attention and cognition, rather than in environmentally stimulated arousal per se, dysfunction of PFC circuits mediating phasic DA release has been invoked to explain behavioral hypersensitivity to environmental stimuli in ADHD (Sikstrom, 2007). This view of ADHD as a disorder of circuits mediating environmentally stimulated arousal suggests that further study of such circuits in humans and in vertebrate animal models, as well as in Drosophila, may improve understanding of this disorder and ultimately lead to improved therapeutics (Lebestky, 2009).


Identification of dDA1 mutants

To identify dDA1 mutants, multiple fly lines with lesions that are known to map at the chromosomal location 88A where the dDA1 gene resides were surveyed. Two lines showed abnormal dDA1 immunoreactivities in the brain. One of them is the inversion line In(3LR)234, which has the break points at 67D and 88A-88B. The other is f02676 containing the transposable element piggyBac inserted at the fimmunoreactivitiest intron in the dDA1 locus. dDA1 is highly enriched in the MB lobes, the central complex, a few scattered cells in the brain, and the Apterous-positive cells in the thoracico-abdominal ganglion (Kim, 2003; Park, 2004). Both In(3LR)234 and f02676 have negligible dDA1 immunoreactivities in the MB and the central complex but intact immunoreactivities in the scattered and Apterous-positive cells. Consistently, full-length dDA1 transcripts were detected in both lines by RT-PCR. Thus, In(3LR)234 and f02676 appear to have lesions in the regulatory sequence for tissue-specific dDA1 expression, representing hypomorphic dDA1 alleles, and are designated as dumb1 [D1(uno) in mushroom bodies] and dumb2, respectively (Kim, 2007b).

Impaired learning of dumb mutants in aversive conditioning

The observations that dDA1 is concentrated in the MB neuropil and can activate the cAMP pathway (Sugamori, 1995; Kim, 2003) prompted an investigation of the role of dDA1 in olfactory conditioning. When subjected to aversive conditioning using odorants octanol (OCT) and benzaldehyde (BA) as conditioned stimuli and electric shock as a US, dumb1 homozygous mutants showed severely impaired performance immediately after training. Performance of dumb1 did not decline at 1 h after training, suggesting that dumb1 is defective in learning rather than memory. dumb1 has two break points caused by inversion. Thus, to investigate the lesion accountable for poor performance of dumb1, two deficiency lines were used, Df(3L)AC1 and Df(3R)su(Hw)7, having deletion between chromosomes 67A2 and 67D13 and chromosomes 88A9 and 88B2, respectively, which include each break point. Similar to dumb1, dumb1/Df(3R)su(Hw)7 trans-heterozygous mutants exhibited poor performance immediately or 1 h after training. In contrast, performance of dumb1/Df(3L)AC1 was comparable to that of Canton-S and dumb1/+ or Df(3R)su(Hw)7/+ heterozygous flies immediately after training. These data indicate that the lesion in chromosome 88A is responsible for poor learning of dumb1 mutants. The flies heterozygous for both deficiency chromosomes had slightly lower performance scores compared with those of Canton-S and dumb1/+ at 1 h after training. This could be attributable to putative memory genes in the deleted chromosomes (Kim, 2007b).

It was next asked whether the dumb1 phenotype is linked to the lesion in dDA1 by examining the independent dumb allele dumb2 and dumb1/dumb2 trans-heterozygous mutants in aversive conditioning. Like dumb1, both genotypes had negligible performance scores immediately after or 1 h after training, supporting the potential role of dDA1 in punishment-mediated olfactory learning. dumb1 and dumb2 heterozygous flies exhibited normal performance; thus, a single copy of dDA1 may be sufficient for mediating this process (Kim, 2007b).

To test whether dumb mutants could learn better with different conditioned stimuli, other odorants were used in electric shock-mediated olfactory conditioning. When trained with ethyl acetate (EA) and isoamyl acetate (IAA) as conditioned stimuli, dumb1 mutants also displayed severely impaired learning. This suggests that dDA1 is involved in aversive learning induced by diverse odor inputs. The impaired performance of dumb mutants is not attributable to anomalous sensory modalities because all dumb alleles and the control Canton-S and w1118 flies showed comparable avoidance of the CS odors and electric shock presented at two different concentrations or intensities, respectively. Thus, poor learning of dumb mutants is likely attributable to their inability to associate CS+ with US (Kim, 2007b).

Synaptic output of dopamine neurons has been shown to be required during training for aversive learning (Schwaerzel, 2003), implicating the similar requirement of dDA1 at the time of learning. To test this, the pan-neuronal driver Elav-GAL4 and GAL80ts, which allows the temporal control of GAL4 activities, was used. GAL80 binds to GAL4 to sequester it from activating upstream activating sequence (UAS). The temperature-sensitive GAL80ts can no longer bind to GAL4 at 30°C, allowing it to act on UAS to induce downstream gene expression. The piggyBac inserted at the first intron of the dDA1 gene in dumb2 has UAS. Although the piggyBac insertion itself interferes with endogenous dDA1 expression in dumb2, UAS in piggyBac, after binding to GAL4, may induce dDA1 transcription from the second exon containing the 5' untranslated sequence and the start codon. Thus, dumb2 was crossed with dumb1 carrying Elav-GAL4 and GAL80ts to generate Elav-GAL4,GAL80ts/+;dumb1/dumb2 flies. The Elav-GAL4,GAL80ts/+;dumb1/dumb2 kept at room temperature did not have any detectable dDA1 induction; however, when the flies were reared at 30°C for 3 d, conspicuous dDA1 IR was visible in the MB lobes and pedunculi, the central complex, and other brain areas including antennal lobes. Whereas Elav-GAL4 is expressed in all neurons, membrane-bound GFP reporters driven by Elav-GAL4 are enriched in certain brain areas including the aforementioned structures. Therefore, the temporal manipulation of GAL80ts and Elav-GAL4 activities was effective in restricting dDA1 expression at the adult stage in dumb mutants (Kim, 2007b).

When Elav-GAL4,GAL80ts/+;dumb1/dumb2 flies reared at room temperature were subjected to electric shock-mediated conditioning, they showed poor learning; however, their performance was dramatically improved after temperature shift to 30°C. The performance score of Elav-GAL4,GAL80ts/+;dumb1/dumb2 with the restored dDA1 expression was slightly lower than that of Canton-S; nonetheless, it was not significantly different from that of Canton-S treated with the same temperature shift but was different from that of uninduced Elav-GAL4,GAL80ts/+;dumb1/dumb2 (p = 0.0009). Therefore, dDA1 is required in the adult neurons, presumably at the time of training, for aversive memory formation. Notably, the same manipulation in the dumb2 heterozygous background (Elav-GAL4,GAL80ts/+;dumb2/+) did not alter the performance scores after brief training (2 pulses of electric shock) or regular training (12 pulses of electric shock). This indicates that the ectopically expressed dDA1 has a negligible effect on normal learning of the heterozygous flies and thus unlikely contribute to the reinstated performance of dumb1/dumb2 mutants (Kim, 2007b).

It was next asked whether the learning phenotype of dumb mutants is attributable to deficient dDA1 function in the MB rather than in the central complex or other neurons. MB247-GAL4 contains 247 bp of dMEF2 regulatory sequence that allows GAL4 expression rather specifically in a subset of the MB neurons projecting to the α/ß lobes and the gamma lobes, but not the α'/ß' lobes. When MB247-GAL4/UAS-GFP in the wild-type background was stained with the dDA1 antibody, the GFP-labeled (thus MB247-GAL4-expressing) MB neurons were positive for dDA1 immunoreactivities although the relative intensities of GFP and dDA1 signals varied in the different MB lobes. Thus, MB247-GAL4 was used to reinstate dDA1 expression in the MB of dumb mutants. After staining with anti-dDA1 antibody, dDA1 expression was apparent in the MB lobes and pedunculi but not in other neural structures of MB247-GAL4/+;dumb1/dumb2. When subjected to electric shock-mediated conditioning, MB247-GAL4/+;dumb1/dumb2 or MB247-GAL4/+;dumb2/dumb2 had the learning scores comparable to those of Canton-S. Moreover, fully reinstated performance was observed in MB247-GAL4/+;GAL80ts,dumb2/dumb1 reared at 30°C for 3 d before training but not in MB247-GAL4/+;GAL80ts,dumb2/dumb1 reared at room temperature. Therefore, dDA1 expressed only in the subset of the adult MB neurons is necessary and sufficient to rescue the dumb mutant's impaired learning, indicating the indispensable role of the MB dDA1 in aversive memory formation (Kim, 2007b).

Appetitive learning requires dDA1 in the MB

Dopamine is crucial in appetitive learning in mammals; however, the previous study (Schwaerzel, 2003) of TH-GAL4/UAS-Shits flies suggests that this is not the case in Drosophila. To investigate this further, dumb mutants were tested in sugar-mediated olfactory conditioning. Surprisingly, both dumb1 and dumb2 mutants exhibited poor performance immediately after training. Although dumb mutants' performance in appetitive learning was not as severely impaired as in aversive conditioning, it was significantly different from that of Canton-S. As in electric shock-mediated conditioning, dumb mutants' performance did not decline at 1 h after training, indicating a crucial role of dDA1 in acquisition, as opposed to short-term memory, of appetitive conditioning. Moreover, dumb2 homozygous or dumb1/dumb2 trans-heterozygous mutants carrying MB247-GAL4 displayed fully reinstated learning in sugar-mediated conditioning. These data indicate that dDA1 is required in the same subset of the MB neurons for aversive and appetitive learning (Kim, 2007b).

Drosophila D1 dopamine receptor mediates caffeine-induced arousal

The arousing and motor-activating effects of psychostimulants are mediated by multiple systems. In Drosophila, dopaminergic transmission is involved in mediating the arousing effects of methamphetamine, although the neuronal mechanisms of caffeine (CAFF)-induced wakefulness remain unexplored. This study shows that in Drosophila, as in mammals, the wake-promoting effect of CAFF involves both the adenosinergic and dopaminergic systems. By measuring behavioral responses in mutant and transgenic flies exposed to different drug-feeding regimens, it was shown that CAFF-induced wakefulness requires the Drosophila D1 dopamine receptor (dDA1) in the mushroom bodies. In WT flies, CAFF exposure leads to downregulation of dDA1 expression, whereas the transgenic overexpression of dDA1 leads to CAFF resistance. The wake-promoting effects of methamphetamine require a functional dopamine transporter as well as the dDA1, and they engage brain areas in addition to the mushroom bodies (Andretic, 2008).

In Drosophila, the wake-promoting action of the adenosinergic antagonist CAFF is mediated through the dDA1 receptor. Genetic manipulations of the dDA1 receptor, as in dumb1 mutants, or overexpression of dDA1 in the MBs of transgenic flies both lead to resistance to the arousing effects of CAFF. These apparently paradoxical findings can be reconciled if the CAFF response requires downregulation of the dDA1 receptor in the MBs within a certain range. In support of this model, the dDA1 mRNA transcript in WT flies is downregulated in response to either short-term exposure (STE) or long-term exposure (LTE) to CAFF, the dDA1 product is already reduced to a negligible level in the MBs (and most other regions) of the dumb mutant, and excess expression of the dDA1 receptor in the MBs produces CAFF resistance, suggesting that levels in these flies cannot be sufficiently downregulated (Andretic, 2008).

MBs are thought to play a role in the control of arousal. MBs have an inhibitory effect on locomotor activity but a stimulatory effect toward sleep. Genetic and transgenic manipulations of MBs, which lead to decreasing amounts of sleep, are often accompanied by a shortening of sleep episodes, and can thus be explained by a premature arousing signal (Andretic, 2008).

The observation that the doses of CAFF that decrease sleep also increase motor activity is similar to the effect of CAFF in vertebrates. In mammals, the antagonistic effect of CAFF on adenosine receptors located on dopaminergic neurons leads to increased release (see Solinas, 2002). A similar mechanism might be operating in flies, based on the correlation between CAFF responsiveness and functional dDA1 receptors in MBs as well as on the motor-activating effects of dopamine (Andretic, 2008).

Although CAFF and methamphetamine (METH) lead to similar wake-promoting and motor-activating effects, the neuronal mechanisms underlying responses to these drugs are only partially overlapping. Both responses require a functional dDA1 receptor, particularly in the MBs, but METH does not lead to uniform downregulation of dDA1 in the brain, although it is conceivable that downregulation might occur in a limited area of the brain outside of the MB. Although CAFF-induced wakefulness involves dDA1 downregulation in MBs, METH-induced wakefulness could involve a selective increase of dDA1 in MBs, whereas dDA1 expression might be unchanged or even decreased in other brain areas. Such an interpretation is supported by the lack of significant modulation of dDA1 transcript in samples obtained from the entire brain of METH-fed flies as well as weaker rescue of METH response when dDA1 was expressed in the entire brain vs. the MBs. When dDA1 expression is restricted only to areas outside of the MBs, METH response is at least as great as in panneural (elav) expression, further suggesting the possibility of antagonism between MBs and other areas for this effect. Another DA receptor, damb, which is specific to the MBs, is not relevant to these responses. It does not show altered regulation in response to CAFF or METH in WT or dumb mutants, and dDA1 expression alone or in combination with CAFF or METH is not altered in damb mutants (Andretic, 2008).

Altogether, these findings suggest a model in which the arousing and motor-activating effects of CAFF are a consequence of its neuromodulatory action on dopaminergic signaling. This is based on similar behavioral responses to CAFF and CPT, a stimulant drug, in Drosophila, which implies that the arousing properties of CAFF involve close interaction between the adenosine and dopamine systems, as they do in mammals. Presynaptically, CAFF can increase dopamine release by antagonizing adenosine receptors on dopaminergic neurons. Resistance to the wake-promoting effect of the A1R antagonist in dumb1 mutants and decreased expression of dDA1 in WT flies after CAFF exposure support a model in which the adenosinergic system acts as a neuromodulator of dopaminergic signaling. CAFF acting through AdoR on dopaminergic neurons could stimulate dopamine synthesis or release through protein kinase A dependent mechanisms similar to the A2A receptor in mammals. Postsynaptically, dDA1 receptors located on MB neurons respond homeostatically by downregulating their expression, a common adaptive mechanism in response to excessive stimulation. A related mechanism involving A1-D1 receptor interaction was observed in the rodent brain and implicated in the psychostimulant properties of CAFF. Furthermore, a recent Drosophila report shows increased dopaminergic content concomitant with decreased dDA1 expression in the brains of sleep-deprived flies (Andretic, 2008).

Although the function of sleep still remains a mystery, one line of evidence suggests that synaptic plasticity underlying memory consolidation might occur during sleep. That such a conserved function of sleep might be present in Drosophila has been sparked by a number of recent reports showing overlap between genes [dunce, rutabaga, Clock, Shaker, 5HT1A, and GABA(A)] and anatomical regions (MBs), which regulate sleep as well as learning and memory. The current findings show that dDA1, a receptor with a role in neuronal plasticity in MB-dependent learning tasks, has only a moderate role in regulation of baseline sleep, although it is important in conditions of elevated arousal, such as those induced by stimulants (Andretic, 2008).

Optimal behavioral performance, such as learning, is dependent on adequate levels of arousal. Although psychostimulant exposure increases dopaminergic transmission and increases general arousal, it also influences specific functions related to reward. These multiple roles are preserved in Drosophila, in which mechanisms for arousal and learning converge on the dDA1 receptor, thus ensuring that learning associated with survival occurs in an attentive and awake organism. CAFF and METH effects on dDA1 receptors in MBs could be mimicking, albeit at an elevated level, the increased dopaminergic signaling that otherwise occurs during learning and memory, reflecting the role that dDA1 receptors play in that process (Andretic, 2008).

D1 receptor activation in the mushroom bodies rescues sleep-loss-induced learning impairments in Drosophila

Extended wakefulness disrupts acquisition of short-term memories in mammals. However, the underlying molecular mechanisms triggered by extended waking and restored by sleep are unknown. Moreover, the neuronal circuits that depend on sleep for optimal learning remain unidentified. In this study learning was evaluated with aversive phototaxic suppression. In this task, flies learn to avoid light that is paired with an aversive stimulus (quinine-humidity). Extensive homology is demonstrated in sleep-deprivation-induced learning impairment between flies and humans. Both 6 hr and 12 hr of sleep deprivation are sufficient to impair learning in Canton-S (Cs) flies. Moreover, learning is impaired at the end of the normal waking day in direct correlation with time spent awake. Mechanistic studies indicate that this task requires intact mushroom bodies (MBs) and requires the dopamine D1-like receptor (dDA1). Importantly, sleep-deprivation-induced learning impairments could be rescued by targeted gene expression of the dDA1 receptor to the MBs. These data provide direct evidence that extended wakefulness disrupts learning in Drosophila. These results demonstrate that it is possible to prevent the effects of sleep deprivation by targeting a single neuronal structure and identify cellular and molecular targets adversely affected by extended waking in a genetically tractable model organism (Seugnet, 2008).

Sleep-deprivation-induced learning impairments were evaluated via an assay that requires flies to inhibit a prepotent attraction toward light. In this task, flies are placed in a T maze and allowed to choose between a lighted and a dark chamber. Filter paper is wetted with 10-1M quinine hydrochloride solution and placed into the lighted chamber such that the quinine and the humidity provide an aversive stimulus. The percentage of times the fly visits the dark vial is tabulated during 16 trials. Flies learn to select the dark alley more frequently over the course of the 16 trials. Learning reaches a maximum during the last four trials of the test and does not improve with additional training. Thus, the performance index is calculated as the percentage of times the fly chooses the dark vial during the last four trials. The assay will be referred to as aversive phototaxic suppression (APS) (Seugnet, 2008).

Flies, like humans, are awake during the day and consolidate their sleep during the night. Canton-S (Cs) flies exhibit a sleep rebound after 3 hr, 6 hr, and 12 hr of sleep deprivation. This study shows that 6 hr and 12 hr of sleep deprivation disrupt learning. Low motivation is an unlikely explanation for the impairment because the time to complete the 16 trials (TCT) was not significantly different from that of controls. Similarly, after sleep deprivation, male flies maintained motivation to court virgin females, another prepotent response, and were not different from controls. Sleep deprivation does not alter the photosensitivity index (PI; percentage of photopositive choices in the T maze in ten trials in the absence of quinine-humidity) nor the quinine-sensitivity index (QSI; time in seconds flies reside on the nonquinine side of a chamber), indicating that the learning impairment is due to sleep loss and not due to sleep-deprivation-induced alterations in sensory thresholds. Indeed, sleep deprivation does not alter photosensitivity when measured over a range of light intensities, nor does it change performance with a fast phototaxis assay. Because flies must climb upward to enter either chamber, the effects of sleep deprivation on geotaxis were evaluated; it was found to be unaffected by sleep deprivation. Importantly, flies that have been selected to prefer climbing downward with gravity learn as well as flies that have been selected to prefer climbing upward against gravity, indicating that geotaxis is not required in this assay. Together, these data indicate that the effects of extended waking are not due to changes in sensory thresholds (Seugnet, 2008).

To determine whether the decrement in performance was the consequence of the stimulus used to keep the animal awake rather than sleep loss per se, several control experiments were conducted. First, flies wee exposed to the perturbations induced by the apparatus for 6 hr between zeitgeber time ZT0 and ZT5:59. Keeping flies awake during this time does not result in subsequent changes in sleep. As expected, exposure to the stimulus in the absence of sleep loss did not result in an additional learning deficit. Currently, all studies that have kept flies awake, including sleep deprivation by gentle handling, have used methods that share common features. To exclude the possibility that these methods impair performance, a novel sleep-deprivation apparatus was invented. The sleep-interrupting device (SLIDE) consists of a thin plastic floor inserted into the tubes underneath flies that can be manipulated like a treadmill. When flies are kept awake with this approach, learning is impaired (Seugnet, 2008).

Sleep fragmentation in humans and rodents is associated with learning impairments. To determine whether sleep fragmentation also deteriorates learning in flies, advantage was taken of the observation that ~10%-15% of Cs flies spontaneously exhibit fragmented sleep while maintaining normal total sleep time. Learning was impaired in flies with fragmented sleep compared to their siblings with consolidated sleep. Thus, even in the absence of mechanical stimulation, sleep fragmentation is associated with learning impairments. Flies with consolidated and fragmented sleep displayed similar control metrics, indicating that they did not differ in sensory thresholds or motoric ability. Importantly, experimentally induced sleep fragmentation impairs learning in otherwise sleep-consolidated flies indicating that sleep fragmentation impairs learning in flies as it does in humans (Seugnet, 2008).

Performance decrements observed in sleep-deprived humans have, at times, been attributed to the intrusion of sleep into periods of waking rather than cognitive impairment per se. Are the learning impairments in flies simply due to high sleep drive? To test this hypothesis, a protocol was designed that allowed separation of the effects of extended wakefulness from increased sleepiness. When flies are deprived of sleep for 22 hr and released into recovery in the evening, sleep rebound is only observed the following morning. If sleep drive impairs performance, flies released into recovery at night should show a deficit when tested the next morning. Flies with high sleep drive exhibit normal performance, indicating that the amount of prior waking rather than interference due to sleepiness is responsible for learning deficits (Seugnet, 2008).

Is a full night of sleep required to restore learning? Performance after sleep deprivation was restored to the baseline level when flies were allowed to nap for 2 hr. In contrast to spontaneous daytime sleep, which is characterized by short sleep bouts, the naps following sleep deprivation resemble nighttime sleep. Thus, as in humans, naps improve learning in flies. Environmental and social factors can alter motivation and temporarily reduce the negative impact of sleep deprivation on performance. For example, sleep-deprived subjects who were given a monetary reward for correct responses were able to maintain performance longer than controls. To evaluate this relationship in flies, the assay was modified by placing a piece of dry filter paper previously soaked in a sucrose solution in the dark vial. Under baseline conditions, the presence of sucrose did not alter performance. However, after 12 hr of sleep deprivation, flies tested with dry sucrose in the dark alley performed as well as flies that had obtained a full nights' sleep. These beneficial effects were lost when sleep deprivation was extended to 36 hr, indicating that deficits cannot be entirely compensated by motivational factors (Seugnet, 2008).

It has been hypothesized that in humans, neurobehavioral deficits accrue when wake time extends beyond a minimal interval measured in hours. In flies, daytime sleep is characterized by short bouts. Interestingly, learning is highest in the morning and declines as the amount of waking accrues during the biological day. Control metrics are similar over the course of the day, indicating that the decrements in performance cannot be explained by circadian modulation of sensory thresholds. However, circadian factors have been shown to influence learning. Thus sleep deprivation was combined with the napping protocols described above to vary the duration of waking at a given circadian time. Three experimental conditions were used, and in each instance performance was evaluated at ZT4. Performance at ZT4 was dependent upon prior wake duration, suggesting that learning is impaired as a function of time spent awake. Interestingly, daytime sleep appears to be less restorative than consolidated sleep observed during the nap. Because performance is reduced by the end of the day, these data suggest that consolidated sleep is required after each waking day to restore optimal learning. Indeed, learning is restored in the evening after 3 hr of spontaneous sleep (ZT12-ZT15) but remains impaired in circadian matched siblings that were kept awake until ZT15 (Seugnet, 2008).

No neural substrate has been identified for APS. A likely candidate is the mushroom bodies (MBs), given their role in many but not all learning and memory tests. MBs play a role in olfactory memory acquisition and play a role in decision making under conflicting situations. The MBs have recently been shown to regulate sleep and inhibitory control. They can be ablated in the fly by feeding larvae hyroxyurea (HU). Although ablation of the MBs disrupts sleep, a minority of HU flies exhibit normal sleep, thereby allowing determination of whether performance is influenced by the MBs independently of sleep time. Learning is impaired in the absence of MBs in all short- and long-sleeping flies; control metrics were unaffected. HU also results in a reduction of antennal lobe size, raising the possibility that the learning impairment may be due to deficits in olfactory processing. However, smell-blind (sbl-1) flies that are olfactory defective perform as well as Cs flies, indicating that olfactory input is not required in this assay (Seugnet, 2008).

To determine whether sleep-deprivation-induced impairments in learning can be explained through alterations in DA signaling, DA levels were evaluated. Whole-head DA levels are significantly elevated after sleep deprivation and are associated with the transcriptional downregulation of the Drosophila dopamine 1-like receptor (dDA1). Downregulation of dDA1 transcripts is also seen in flies with spontaneously fragmented sleep. The pharmacology of DA agonists has been characterized, and these drugs are known to be biologically active in flies. Ritalin, methamphetamine, L-DOPA, and the D1 agonist SKF82958 rescued performance after sleep deprivation; none of these treatments enhanced learning in baseline conditions. Control metrics were unaffected by pharmacologic manipulations. Thus, global enhancement of dopamine signaling overcomes deficits in learning in flies as it does in humans (Seugnet, 2008).

To determine the extent to which DA is involved in this learning assay, additional genetic and pharmacological experiments were conducted. DA levels were reduced by feeding flies the tyrosine hydroxylase (TH) inhibitor 3-iodo L-tyrosine (3IY). Performance was impaired in flies fed 3IY, and this impairment could be rescued by coadministration of L-DOPA. Consistent with previous reports, 3IY consolidated sleep without reducing the intensity of locomotor activity. PI and QSI were unaffected by drug treatment, whereas flies fed 3IY took significantly longer to complete 16 trials. Although TCT was increased in flies fed 3IY, it was not outside the range seen in Cs flies and thus cannot explain the deficit. In addition, disruption of synaptic output from dopaminergic neurons by expression of a temperature-sensitive allele of shibire (UAS-shits1) also impairs learning. Thus, reduction of DA signaling, with either pharmacology or genetics, impairs performance in APS (Seugnet, 2008).

Although a recent study has shown that the dDA1 receptor is important for Pavlovian conditioning, its role in other learning paradigms is unknown. Because the pharmacology of the four Drosophila DA receptors has been investigated, either a D1 (SCH23390) or D2 (eticlopride) antagonist were administered for 2 hr before evaluating learning. SCH23390 and eticlopride have been shown to activate separate behaviors in flies, and although SCH23390 blocks both D1-like receptors (dDA1 and dopamine receptor in mushroom bodies [DAMB]), eticlopride does not. Both D1 and D2 antagonists modified sleep at this dose, indicating they are biologically active. However, only the D1 antagonist disrupted performance. Importantly, the induction of G?s in flies fed the D1 agonist SKF 82958 was blocked by coadministration of the D1 antagonist (Seugnet, 2008).

Because the D1 agonist and D1 antagonist are active at both the dDA1 and the DAMB receptors, learning was evaluated in flies mutant for dDA1. The dDA1 receptor is heavily expressed in MB neuropile and is required for olfactory learning. dumb2 is a hypomorphic allele that reduces dDA1 expression in the mushroom bodies. The P element insertion PL00420 (dumb3) removes most of dDA1 expression in the MBs while inducing ectopic expression in glia and the optic lobes. Both alleles have reduced learning. dumb2 and dumb3 mutants exhibited normal sleep, PI, and QSI, but dumb3 flies had 12% longer TCT. To confirm that this phenotype maps to the dDA1 locus, mutant flies were crossed with flies carrying a deficiency (Df) of the dDA1 locus, Df(3R)red1. Learning was significantly reduced in the resulting dumb2/Df and dumb3/Df flies, indicating that the impairments were due to disruption of dDA1 expression. Finally, the D1 agonist SKF 82958 was administered to mutant flies, and learning was assessed. Performance could not be rescued by the D1 agonist. Because the D1 agonist did not restore learning in either the mutants, it is unlikely that the previous improvement in learning after sleep deprivation was due to nonspecific effects of SKF82958 at other receptors (Seugnet, 2008).

Imaging studies in humans suggest that performance decrements after waking may not be due to global brain impairments and thus may reflect a molecular vulnerability in specific neuronal circuits. To determine whether waking impairs learning by modifying dDA1 globally or in specific circuits, dDA1 was manipulated only in the MBs. The piggyBac inserted into the first intron of the dDA1 gene in the dumb2 mutants contains a UAS that can be used to induce functional dDA1 receptor. The gene-switch system (MBSwitch) was used to avoid potential developmental defects. MB-Switch/+; dumb2/+ flies fed RU486 maintained learning after extended wakefulness, whereas their vehicle-fed siblings were impaired. Interestingly, RU486-treated MB-Switch/+; dumb2/+ had no effect on baseline learning in the absence of sleep loss, and baseline sleep was not altered. As expected, the parental lines learn normally when exposed to RU486 and are impaired after extended waking (Figure 6G). Furthermore, RU486 has no effect on learning in Cs flies, either under baseline condition or during extended waking (Seugnet, 2008).

These data provide direct evidence that extended waking disrupts learning and is amenable to genetic dissection in Drosophila. Importantly, manipulation of dDA1 only in the MBs, which represent ~2% of the total number of neurons in the Drosophila central nervous system, was sufficient to prevent the learning deficits associated with extended waking. These data support the hypothesis that extended waking can deteriorate the function of specific brain areas that are critical for adaptive behavior (Seugnet, 2008). Sleep-deprivation experiments are inherently problematic in that it is frequently difficult to determine whether an observed outcome is because of the lack of sleep or the methods used to keep the organism awake. Thus, several control experiments were conducted to evaluate potential confounding variables. It was found that although learning is disrupted when extended waking is achieved by mechanical stimulation, mechanical stimulation in the absence of sleep loss produced no deficits in learning. Importantly, spontaneous waking and sleep fragmentation impair learning without mechanical stimulation. Together, these data indicate that it is the extended waking per se that disrupts learning (Seugnet, 2008).

In Drosophila, dopaminergic neurons project arborizations to the MB neuropile, where they influence aversive learning. Although a recent study has shown that the dDA1 receptor is important for olfactory conditioning, its role in other learning paradigms is unknown. The current results extend the role of dDA1 receptor beyond olfactory learning. It is worth noting that a role for D1 receptor in short-term memory and response inhibition has been reported in humans, nonhuman primates, and rodents. Previous studies have shown that DA in the MBs plays a role in decision making under conflicting situations and may signal the aversive stimulus to the MBs in olfactory conditioning. Interestingly, flies in the APS also face a conflicting choice between their prepotent attraction toward light and the aversive stimulus. Thus, the modulation of DA signaling observed during extended waking may disrupt performance by multiple mechanisms. Interestingly, children with Attention Deficit Hyperactivity Disorder exhibit both disorganized DA signaling and difficulty with response inhibition. Moreover, sleep problems are highly prevalent in ADHD and, when present, are associated with poorer child outcomes (Seugnet, 2008).

In conclusion, sleep deprivation impairs short-term memory and response inhibition in Drosophila. The data demonstrate that waking is particularly deleterious for DA circuits that are crucial for maintaining adaptive behavior. Because optimal performance can only occur within a narrow range of DA signaling and DA signaling is easily disrupted by waking, it is proposed that an important role of sleep may be to restore DA homeostasis. Nonetheless, it is likely that sleep loss impacts the brain by altering a number of molecular pathways. Together, these experiments pave the way for the identification of the underlying molecular mechanisms (Seugnet, 2008).

Dopamine neurons modulate pheromone responses in Drosophila courtship learning

Learning through trial-and-error interactions allows animals to adapt innate behavioural 'rules of thumb' to the local environment, improving their prospects for survival and reproduction. Naive Drosophila melanogaster males, for example, court both virgin and mated females, but learn through experience to selectively suppress futile courtship towards females that have already mated. This study shows that courtship learning reflects an enhanced response to the male pheromone cis-vaccenyl acetate (cVA), which is deposited on females during mating and thus distinguishes mated females from virgins. Dissociation experiments suggest a simple learning rule in which unsuccessful courtship enhances sensitivity to cVA. The learning experience can be mimicked by artificial activation of dopaminergic neurons, and this study identified a specific class of dopaminergic neuron that is critical for courtship learning. These neurons provide input to the mushroom body (MB) γ lobe, and the DopR1 dopamine receptor is required in MBγ neurons for both natural and artificial courtship learning. This work thus reveals critical behavioural, cellular and molecular components of the learning rule by which Drosophila adjusts its innate mating strategy according to experience (Keleman, 2012).

Mature virgin Drosophila females are usually willing to mate, whereas those that have recently mated are generally recalcitrant to further mating attempts. A male thus increases his overall mating success if he concentrates his courtship efforts on virgins. Given geographic and seasonal fluctuations in the relative abundance of virgins and mated females, and the cues that distinguish them, the optimal courtship strategy is unlikely to be a species universal. A heuristic for approaching this optimum could, however, be universal, allowing evolution to select for genes that implement such a learning rule in the fly's brain (Keleman, 2012).

A male's courtship behaviour can be quantified by a courtship index (CI), and his ability to discriminate virgins from mated females by a discrimination index (DI), the relative reduction in the mean CI in single-pair assays with mated versus virgin females: DI = [CIv-CIm]/CIv. In courtship assays, naive males courted mated females only marginally less vigorously than they courted virgins, whereas males that had experienced rejection from mated females were subsequently much less active when courting mated females than virgins. The relative difference between the mean CIs of experienced (CI+) and naive (CI) males gives rise to a learning index: LI = [CI-CI+<]/CI. For males trained with mated females, the LI was just 7.8% in tests with virgin females but 48.2% when tested with mated females. Similar results were obtained when decapitated virgins were used as trainers, suggesting that male behaviour is conditioned by the failure to mate, not by active rejection from the female (Keleman, 2012).

To discriminate mated females from virgins, a male might detect either the subtle changes in female pheromones on mating or the telltale vestiges of male pheromones that linger on mated females. The male-specific pheromone cVA is transferred to the female cuticle on mating. It is not detectable on the cuticle of either males or virgin females. Naive Or67d mutant males, which are unable to detect cVA, courted virgin and mated females equally (DI = −0.4%) and did not benefit from training. In contrast, analogous mutations in either of two other candidate pheromone receptor genes, Or47b and Gr68a, did not impair discrimination or learning. cVA detection is therefore crucial for naive and experienced males to discriminate mated females from virgins (Keleman, 2012).

The salient feature of training might be the presence of cVA on the mated female, the lack of courtship success, or an association formed between the two. A dissociation experiment was designed to distinguish between these possibilities. Female post-mating behaviour, including courtship rejection, is triggered by sex peptide (SP), a male seminal fluid peptide transferred to the female during mating. Virgin females in which SP is transgenically expressed in the nervous system reject courting males (pseudomated females), whereas females that have mated with SP-null mutant males are still receptive (pseudovirgins). As expected, cVA was detected on the cuticle of both mated females and pseudovirgins, but not on virgins or pseudomated females. Thus, with pseudomated and pseudovirgin females the presence of cVA and sexual receptivity are fully dissociated (Keleman, 2012).

Pseudomated females were just as effective as genuinely mated females when used as trainers, whereas pseudovirgin females were not. In contrast, pseudovirgin but not pseudomated females were as effective as mated females when used as testers. Indeed, robust courtship learning was observed when males were trained with pseudomated females and tested with pseudovirgins, but not vice versa. It is therefore concluded that the salient feature of training is simply the lack of courtship success, not its association with cVA, and that training alters the male’s response to cVA or some other vestige of previous contact with another male (Keleman, 2012).

To test whether training does indeed alter sensitivity to cVA, varying doses of cVA were applied to pseudomated females and presented as testers to naive and experienced males. As expected, high doses of cVA inhibited courtship by both naive and experienced males. However, males trained with either mated or pseudomated females were inhibited by much lower doses of cVA than naive males were. Courtship training did not enhance sensitivity to an unrelated aversive odorant (Keleman, 2012).

Dopamine is thought to provide a learning signal in a variety of different models and species, including aversive olfactory learning and conditioned suppression of male–male courtship in Drosophila. If dopamine also encodes an instructive signal during courtship learning, then artificial stimulation of dopaminergic neurons might mimic training with a mated female. To test this, the warmth-activated TrpA1 channel was expressed in most dopaminergic neurons, and attempts were made to'train' naive isolated males by warming them briefly to 30°C. When subsequently returned to 25°C and tested with mated females, the courtship activity of these males was indeed markedly reduced in comparison with that of control males. This suppression was specific for courtship towards mated but not virgin females, was dependent on a functional Or67d receptor, and was correlated with an increased sensitivity to cVA. In these respects, activation of dopaminergic neurons thus mimics a specific courtship learning signal rather than a non-specific punishment signal that might be expected to suppress courtship more generally. Experiments in which various subsets of dopaminergic neurons further suggest that the neurons involved in courtship learning are distinct from those previously implicated in various forms of aversive olfactory learning were selectively activated (Keleman, 2012).

Many aspects of male courtship behaviour have been linked to the set of neurons that express the fruitless (fru) gene . Among these are the Or67d olfactory neurons (OSNs) and MBγ neurons, both of which function in courtship learning. It was speculated that the dopaminergic neurons involved in courtship learning might also be fru+. To test this hypothesis synaptic transmission of fru+ dopaminergic neurons was acutely blocked by using shits, which inhibits synaptic vesicle recycling at 30°C but not at 22°C. Such males showed significantly impaired learning when trained at 30°C and tested at 22°C, but not vice versa. These data thus establish a requirement for dopaminergic neurons in memory formation, not recall, and further indicate that the relevant cells are fru+ (Keleman, 2012).

Previous studies identified two distinct classes of fru+ dopaminergic neurons: aSP4 and aSP13. To test whether aSP4 and/or aSP13 neurons contribute to courtship learning, synaptic transmission was chronically inhibited in these neurons with tetanus toxin light chain (TNT), using drivers selective for either aSP4 or aSP13. With each of five independent aSP13 drivers, learning was reduced by about 50% compared with control males that carried an inactive version of the TNT transgene in the same genetic background. A similar learning deficit was observed in positive controls in which TNT was targeted to both aSP13 and aSP4, to Or67d+ OSNs, or to MBγ neurons. In contrast, courtship learning was unimpaired in assays using either of two driver lines expressed in aSP4 but not aSP13. It is concluded that synaptic transmission of aSP13 neurons is crucial for courtship learning (Keleman, 2012).

The presynaptic termini of aSP13 neurons are located at the tip of the MB γ lobe, indicating that they might convey a dopamine learning signal to MBγ neurons. If so, then a dopamine receptor should be required specifically in MBγ neurons for courtship learning. DopR1 and DopR2 receptors were considered as candidates, and homologous recombination was used to generate analogous loss-of-function alleles for each gene (DopR1attP and DopR2attP, respectively). Both mutants are viable and fertile and homozygous naive males court at normal levels. However, courtship learning was significantly impaired in DopR1attP but not DopR2attP mutants, as was 'fictive learning' induced by thermogenetic activation of dopaminergic neurons. Nevertheless, learning was not completely eliminated in these DopR1 mutants, indicating that other dopamine receptors might also contribute. To confirm that the learning deficit in the DopR1attP mutant was indeed due to loss of DopR1 function, the deleted genomic region was reintegrated by site-specific transgenesis. Males homozygous for this repaired DopR1 allele, DopR1Res, performed just as well as wild-type males in courtship learning assays (Keleman, 2012).

Finally, RNA-mediated interference (RNAi) knockdown and rescue experiments were performed to test whether DopR1 function is indeed required in MBγ neurons. Expression of a DopR1 RNAi transgene selectively in MBγ neurons significantly reduced DopR1 expression levels in the γ lobe and impaired courtship learning. Conversely, the learning disability of DopR1attP mutants was fully alleviated by expressing a DopR1 transgene specifically in MBγ neurons. It is therefore postulate that DopR1 acts in MBγ neurons to transduce a dopamine learning signal provided by aSP13 neurons (Keleman, 2012).

To maximize his reproductive success, a Drosophila male should be highly attuned to those cues that discriminate receptive from unreceptive females. A male that is too selective may miss mating opportunities; a male that is too promiscuous may waste resources on futile courtship. The optimal tuning is likely to vary from place to place and from time to time, depending for example on local and seasonal fluctuations in the abundance and quality of mating partners and the pheromone signals that they provide. This study defines a simple heuristic that could allow the male to learn an effective courtship strategy in his local environment: be promiscuous at first, but become more selective if a mating attempt fails. Furthermore, this study has identified key elements that implement this learning rule in the fly's brain. It is proposed that, when a mating attempt fails, aSP13 dopaminergic neurons convey a learning signal to MBγ neurons through the DopR1 receptor, and that this induces lasting changes in the internal processing of the cVA signal that discriminates mated females from virgins. Further studies of this genetically defined and tractable circuit should provide a detailed understanding of how a relatively simple learning circuit, embedded within decision-making centres of the brain, endows plasticity on an innate behaviour (Keleman, 2012).

Identification of a dopamine pathway that regulates sleep and arousal in Drosophila

Sleep is required to maintain physiological functions, including memory, and is regulated by monoamines across species. Enhancement of dopamine signals by a mutation in the dopamine transporter (DAT) decreases sleep, but the underlying dopamine circuit responsible for this remains unknown. This study found that the D1 dopamine receptor (DA1, also known as DopR) in the dorsal fan-shaped body (dFSB) mediates the arousal effect of dopamine in Drosophila. The short sleep phenotype of the DAT mutant was completely rescued by an additional mutation in the DA1 gene, but expression of wild-type DA1 in the dFSB restored the short sleep phenotype. Anatomical and physiological connections were found between dopamine neurons and the dFSB neuron. Finally, mosaic analysis with a repressive marker found that a single dopamine neuron projecting to the FSB activates arousal. These results suggest that a local dopamine pathway regulates sleep (Ueno, 2012).

Neurons in the dFSB are involved in dopaminergic sleep regulation in Drosophila an the PPM3-FSB dopamine pathway, which is distinct from that required for memory formation, regulates arousal. A previous study found that the rescue of DA1 mutants outside of the mushroom body using a pan-neuronal GAL4 driver, elav-GAL4, coupled with the mushroom body suppressor MB-GAL80 in DA1dumb1 mutants can recover methamphetamine sensitivity. This suggests that dopamine regulates arousal outside of the mushroom body. Previous findings have shown that DA1 in the PDF neurons (lateral ventral neurons) regulates sleep-wake arousal and that DA1 in the ellipsoid body regulates stress-induced arousal (Ueno, 2012).

A previous study identified PDF neurons that mediate the buffering effects of light on dopamine-induced arousal. However, the current ablation experiments showed that dopamine can elicit strong arousal effects without PDF neurons. The previous report used heterozygous DA1 mutant flies in a wild-type background for most experiments. However, this study used homozygous (null) DA1 mutants, as we found that the heterozygous DA1 mutants crossed with DATfmn showed an almost equivalent short sleep phenotype. Thus, one possible explanation for the difference between the previous study and this one is that heterozygous expression of DA1 in the FSB is sufficient to elicit the arousal effects of dopamine, which is also regulated partly through PDF neurons. Given that DA1 expression in the dFSB neurons alone is sufficient for the majority of the wake-promoting effects of dopamine, the arousal regulating dopamine pathway appears to converge at the FSB (Ueno, 2012).

Activation of the dopamine neurons required for aversive memory formation had little effect on sleep. On the other hand, TrpA1 stimulation in a single FSB-projecting PPM3 dopamine neuron was able to reduce sleep. As the magnitude of the decrease in sleep by the activation of the single PPM3 neuron was smaller than that of most dopamine neurons with TH-GAL4, it is possible that other dopamine neurons, which were not labeled in our MARCM screening, also affect arousal. For example, in the PPL1 cluster, at least five types of projections to the mushroom body and FSB-projecting neurons have been described (Ueno, 2012).

However, in a MARCM experiment, many of the labeled neurons in the PPL1 cluster were seen to project to the alpha lobe of the mushroom body; it is possible that the contribution of other PPL1 neurons were not fully determine. Alternatively, combinatorial activation of the FSB-projecting PPM3 neurons and other dopamine neurons may have a synergistic effect. Further MARCM experiments using various clone induction protocols may help to formulate a more comprehensive characterization of single dopamine neurons. In addition, it is also possible that dopamine neurons that are not labeled by TH-GAL4 also have an arousal effect. It was noticed that TH-GAL4–induced GFP expression and tyrosine hydroxylase staining do not overlap completely, and some clusters, such as the PAM cluster, are covered only partially by TH-Gal4 (Ueno, 2012).

The association between sleep and memory in Drosophila has recently been described in various reports. Other short sleep flies, hyperkinetic and calcineurin knockdown, also suffer from impaired memory. Sleep deprivation has been shown to impair aversive olfactory memory and learned phototaxis suppression. Conversely, memory formation increased sleep duration in normal flies, and this has not been observed in learning-deficient mutants. In the Drosophila brain, the expression of synaptic component proteins decreased during sleep and increased during waking, suggesting that sleep is required for the maintenance of synapses. In contrast, the current data imply a functional dissociation between sleep and memory circuits by different dopamine neurons. These results will provide clues to uncover a possible physiological relationship between them, for example, by the activation of only one to examine the causal relationship (Ueno, 2012).

The FSB has been reported to have a role in visual memory processing, but its involvement in other behaviors is not yet fully understood. A recent report showed that activation of dFSB neurons induces sleep. This suggests that the dopamine signal regulates sleep via control of the neural properties of the dFSB neurons through DA1. In mammals, dopamine signaling via the D1-type dopamine receptor is thought to increase firing probability. However, dopamine signaling via the D1-like receptor DA1 in flies results in the inhibition of neural activity. Although the previous report suggested that the inhibition is independent of protein kinase A, activation of adenylyl cyclase with Gs* in dFSB decreased sleep levels. Taken together, these findings indicate that DA1 activation in the dFSB inhibits neural activity and results in the promotion of wakefulness. It has been reported that sleep induction through the activation of the dFSB neurons promotes memory consolidation after courtship conditioning. However, this study found that DA1 expression in the FSB itself had little effect on aversive olfactory memory. The contradiction between these findings might be a result of the difference in the memory tasks. It is also possible that functional interaction between sleep and memory is implemented downstream of the FSB. Further studies are required to elucidate the causal relationship between sleep and memory (Ueno, 2012).


The role of phosphorylation in D1 dopamine receptor desensitization: evidence for a novel mechanism of arrestin association

Homologous desensitization of D(1) dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, a series of mutated D(1) receptor constructs were created. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). Four truncation mutants were created of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. These data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se (Kim, 2004).

Dopamine receptor expression is regulated by direct interaction with the chaperone protein calnexin

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. The ER chaperone protein calnexin has been identified as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, assays were conducted in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, it was shown that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. These data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs (Free, 2007).

Endocytic recycling signal in the D1 dopamine receptor

A critical event determining the functional consequences of G protein-coupled receptor (GPCR) endocytosis is the molecular sorting of internalized receptors between divergent recycling and degradative membrane pathways. The D1 dopamine receptor recycles rapidly and efficiently to the plasma membrane after agonist-induced endocytosis and is remarkably resistant to proteolytic down-regulation. Whereas the mechanism mediating agonist-induced endocytosis of D1 receptors has been investigated in some detail, little is known about how receptors are sorted after endocytosis. A sequence present in the carboxyl-terminal cytoplasmic domain of the human D1 dopamine receptor has been identified that is specifically required for the efficient recycling of endocytosed receptors back to the plasma membrane. This sequence is distinct from previously identified membrane trafficking signals and is located in a proximal portion of the carboxyl-terminal cytoplasmic domain, in contrast to previously identified GPCR recycling signals present at the distal tip. Nevertheless, fusion of this sequence to the carboxyl terminus of a chimeric mutant delta opioid neuropeptide receptor is sufficient to re-route internalized receptors from lysosomal to recycling membrane pathways, defining this sequence as a bona fide endocytic recycling signal that can function in both proximal and distal locations. These results identify a novel sorting signal controlling the endocytic trafficking itinerary of a physiologically important dopamine receptor, provide the first example of such a sorting signal functioning in a proximal portion of the carboxyl-terminal cytoplasmic domain, and suggest the existence of a diverse array of sorting signals in the GPCR superfamily that mediate subtype-specific regulation of receptors via endocytic membrane trafficking (Vargas, 2004).

Regulation of D1 dopamine receptor trafficking and signaling by caveolin-1

There is accumulating evidence that G protein-coupled receptor signaling is regulated by localization in lipid raft microdomains. The D1 dopamine receptor (D1R) is localized in caveolae, a subset of lipid rafts. Through co-immunoprecipitation and bioluminescence resonance energy transfer assays, it has been demonstrated that this localization is mediated by an interaction between caveolin-1 and D1R in COS7 cells and an isoform selective interaction between D1R and caveolin-1alphain rat brain. The D1R interaction with caveolin-1 requires a putative caveolin binding motif identified in transmembrane domain 7. Agonist stimulation of D1R caused translocation of D1R into caveolin-1 enriched sucrose fractions which was determined to be a result of D1R endocytosis through caveolae. This was found to be PKA-independent and a kinetically slower process than clathrin mediated endocytosis. Site directed mutagenesis of the caveolin binding motif at amino acids F313 and W318 significantly attenuated caveolar endocytosis of D1R. It was also found that these caveolin binding mutants had a diminished capacity to stimulate cAMP production which was determined to be due to constitutive desensitization of these receptors. In contrast, D1Rs had an enhanced ability to maximally generate cAMP in chemically induced caveolae disrupted cells. Taken together, these data suggest that caveolae has an important role in regulating D1R turnover and signaling in brain (Kong. 2007).

Dopamine receptor interaction with GPCR associated sorting protein

After activation, most G protein coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. This study shows that a mutant beta-2 adrenergic receptor and a mutant mu opioid receptor that have previously been described as lacking 'recycling signals' due to mutations in their C-termini, in fact bind to GASP and are targeted for degradation. A mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together these results indicate that alteration of receptors in their C-termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation (Thompson, 2007).

Dopamine receptor heteroligomerization

Evidence is provided for the formation of a novel phospholipase C-mediated calcium signal arising from coactivation of D1 and D2 dopamine receptors. In the present study, robust fluorescence resonance energy transfer showed that these receptors exist in close proximity indicative of D1-D2 receptor heterooligomerization. The close proximity of these receptors within the heterooligomer allowed for cross-phosphorylation of the D2 receptor by selective activation of the D1 receptor. D1-D2 receptor heterooligomers were internalized when the receptors were coactivated by dopamine or either receptor was singly activated by the D1-selective agonist (±)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) or the D2-selective agonist quinpirole. The D2 receptor expressed alone did not internalize after activation by quinpirole except when coexpressed with the D1 receptor. D1-D2 receptor heterooligomerization resulted in an altered level of steady-state cell surface expression compared with D1 and D2 homooligomers, with increased D2 and decreased D1 receptor cell surface density. Together, these results demonstrated that D1 and D2 receptors formed heterooligomeric units with unique cell surface localization, internalization, and transactivation properties that are distinct from that of D1 and D2 receptor homooligomers (So, 2005).

D1-D2 dopamine receptor coupling to rapid G-proteins

A heteromeric D1-D2 dopamine receptor signaling complex in brain is coupled to Gq/11 and requires agonist binding to both receptors for G protein activation and intracellular calcium release. The D1 agonist SKF83959 was identified as a specific agonist for the heteromer that activated Gq/11 by functioning as a full agonist for the D1 receptor and a high-affinity partial agonist for a pertussis toxin-resistant D2 receptor within the complex. Evidence that the D1-D2 signaling complex can be more readily detected in mice that are 8 months in age compared with animals that are 3 months old, suggesting that calcium signaling through the D1-D2 dopamine receptor complex is relevant for function in the postadolescent brain. Activation of Gq/11 through the heteromer increases levels of calcium/calmodulin-dependent protein kinase IIalpha in the nucleus accumbens, unlike activation of Gs/olf-coupled D1 receptors, indicating a mechanism by which D1-D2 dopamine receptor complexes may contribute to synaptic plasticity (Rashid, 2007).

Dopamine receptor mutation and learning

Dopamine is an important neurotransmitter involved in learning and memory including emotional memory. The involvement of dopamine in conditioned fear has been widely documented. However, little is known about the molecular mechanisms that underlie contextual fear conditioning and memory consolidation. To address this issue, dopamine D1-deficient mice (D1-/-) and their wild-type (D1+/+) and heterozygote (D1+/-) siblings were used to assess aversive learning and memory. Two different aspects were quantified of fear responses to an environment where the mice have previously received unsignaled footshocks. Using one-trial step-through passive avoidance and conditioned freezing paradigms, mice were conditioned to receive mild inescapable footshocks then tested for acquisition, retention and extinction of conditioned fear responses 5 min after and up to 45-90 days post-training. No differences were observed among any of the genotypes in the acquisition of passive avoidance response or fear-induced freezing behavior. However, with extended testing, D1-/- mice exhibited prolonged retention and delayed extinction of conditioned fear responses in both tasks, suggesting that D1-/- mice are capable of acquiring aversive learning normally. These findings demonstrate that the dopamine D1 receptor is not important for acquisition or consolidation of aversive learning and memory but has an important role in modulating the extinction of fear memory (El-Ghundi, 2001).

Dopamine receptor and sensory plasticity in C. elegans

Dopamine has been implicated in the modulation of diverse forms of behavioral plasticity, including appetitive learning and addiction. An important challenge is to understand how dopamine's effects at the cellular level alter the properties of neural circuits to modify behavior. In the nematode C. elegans, dopamine modulates habituation of an escape reflex triggered by body touch. In the absence of food, animals habituate more rapidly than in the presence of food; this contextual information about food availability is provided by dopaminergic mechanosensory neurons that sense the presence of bacteria. Dopamine alters habituation kinetics by selectively modulating the touch responses of the anterior-body mechanoreceptors; this modulation involves a D1-like dopamine receptor, a Gq/PLC-beta signaling pathway, and calcium release within the touch neurons. Interestingly, the body touch mechanoreceptors can themselves excite the dopamine neurons, forming a positive feedback loop capable of integrating context and experience to modulate mechanosensory attention (Kindt, 2007).

Dopamine and appetitive conditioning in Aplysia

In a recently developed in vitro analog of appetitive classical conditioning of feeding in Aplysia, the unconditioned stimulus (US) was electrical stimulation of the esophageal nerve (En). This nerve is rich in dopamine (DA)-containing processes, which suggests that DA mediates reinforcement during appetitive conditioning. To test this possibility, methylergonovine was used to antagonize DA receptors. Methylergonovine (1 nM) blocked the pairing-specific increase in fictive feeding that is usually induced by in vitro classical conditioning. The present results and previous observation that methylergonovine also blocks the effects of contingent reinforcement in an in vitro analog of appetitive operant conditioning suggest that DA mediates reinforcement for appetitive associative conditioning of feeding in Aplysia (Reyes, 2005).

Dopamine receptor blocade and learning

The involvement of dopamine (DA) in conditioned taste aversion (CTA) learning was studied with saccharin or sucrose as the conditioned stimulus (CS) and intraperitoneal lithium as the unconditioned stimulus (US). The dopamine D1 antagonist R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390) (12.5-50 microg/kg, s.c.), given 5 min after the CS, impaired the acquisition of CTA in a paradigm consisting of three or a single CS-lithium association. SCH 23390 failed to impair CTA acquisition given 45 min after, 30 min before, or right before the CS. (-)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5a-benzo-(d)-naphtho-(2,1b) azepine (SCH 39166) (12.5-50.0 microg/kg, s.c), a SCH 23390 analog that does not bind to 5HT2 receptors, also impaired CTA. No significant impairment of CTA was obtained after administration of the specific D2/D(3) antagonist raclopride (100 and 300 microg/kg, s.c.). The ability of SCH 23390 to impair CTA learning was confirmed by its ability to reduce the conditional aversive reactions to a gustatory CS (sweet chocolate) as estimated in a taste reactivity paradigm. SCH 39166 impaired CTA also when infused in the nucleus accumbens (NAc) shell 5 min after the CS. No impairment was obtained from the NAc core or from the bed nucleus stria terminalis. The results indicate that D1 receptor blockade impairs CTA learning by disrupting the formation of a short-term memory trace of the gustatory CS and that endogenous dopamine acting on D1 receptors in the NAc shell plays a role in short-term memory processes related to associative gustatory learning (Fenu, 2001; full text of article).

The effects of dopamine (DA) D1 and D2 receptor antagonists on the acquisition and expression of flavor-preferences conditioned by the sweet taste of fructose were examined. Food-restricted rats were trained over eight alternating one-bottle sessions to drink an 8% fructose solution containing one novel flavor (CS+) and a less preferred 0.2% saccharin solution containing a different flavor (CS-). Three groups of rats were treated daily with either vehicle (control group), SCH23390 (200 nmol/kg; D1 group), or raclopride (200 nmol/kg; D2 group) during training. Additional groups of vehicle-treated rats had their daily training intakes matched to that of the D1 and D2 groups. Preferences were assessed in two-bottle tests with the CS+ and CS- flavors presented in 0.2% saccharin solutions following doses of 0, 50, 200, 400, or 800 nmol/kg of either D1 or D2 antagonists. The D1 and D2 groups, unlike the control and yoked-control groups, failed to display a significant CS+ preference in the two-bottle tests following vehicle treatment. In addition, treatment with SCH23390 prior to the two-bottle tests blocked the expression of the CS+ preference in the control groups. Pretest raclopride treatment attenuated the CS+ preference at some dose levels. Raclopride also attenuated the preference for fructose in rats given two-bottle training with the CS+/fructose (CS+/F) and CS-/saccharin (CS-/S) solutions. These findings indicate that D1 and D2 antagonists block flavor-preference conditioning by sweet taste and that D1, and to a lesser extent D2, receptor antagonists attenuate the expression of a previously acquired preference (Baker, 2003).

Previous studies have shown that D1 receptor blockade disrupts and D2 receptor blockade enhances long-term potentiation. These data lead to the prediction that D1 antagonists will attenuate and D2 antagonists will potentiate at least some types of learning. The prediction is difficult to test, however, because disruptions in either D1 or D2 transmission lead to reduced locomotion, exploration, and response execution and are therefore likely to impair learning that requires behavioral responding (including exploration of an environment) during the learning episode. Under a paradigm that minimizes motor requirements, rats were trained to enter a food compartment during pellet presentation. Animals then received tone-food pairings under the influence of D1 antagonist SCH23390 (0, 0.4, 0.8, and 0.16 mg/kg) or D2 antagonist raclopride (0, 0.2, 0.4, and 0.8 mg/kg). An additional group received unpaired presentations of tone and food. On a drug-free test day 24 hr later, animals that had been under the influence of SCH23390 (like animals that had received unpaired presentations of tone and food) showed reduced head entries in response to the tone, whereas animals that had been under the influence of raclopride showed increased head entries in response to the tone compared with vehicle controls. These data demonstrate that, under a conditioned approach paradigm, D1 and D2 family receptor antagonists disrupt and promote learning, respectively, as predicted by the effects of D1 and D2 receptor blockade on neuronal plasticity (Eyny, 2003: full text of article).

Conditioned taste aversion (CTA), is a form of Pavlovian learning wherein a novel flavour is powerfully associated with subsequent feelings of illness, and is afterwards avoided. In rats, pharmacological blockade of dopamine D1 receptors has been reported to prevent the expression of a CTA to the sweet taste of sucrose or saccharine. Genetically modified mice were used to determine whether dopamine D1 receptors are necessary for the expression of a CTA. Food-deprived mice lacking the dopamine D1 receptor (D1r-/-) did not express a LiCl-induced (125 or 254 mg/kg) CTA to the sweet taste of 0.5 m sucrose, in agreement with previous pharmacological studies. However, water-deprived D1r-/- mice did express normal LiCl-induced (40, 150 and 254 mg/kg) CTA to a salty taste (0.2 m NaCl). These results suggest that activation of D1 receptors might contribute to the strength of an aversive gustatory association, but might not be required for the formation of a CTA in general (Cannon, 2005).

Recent research has implicated the nucleus accumbens (NAc) in consolidating recently acquired goal-directed appetitive memories, including spatial learning and other instrumental processes. However, an important but unresolved issue is whether this forebrain structure also contributes to the consolidation of fundamental forms of appetitive learning acquired by Pavlovian associative processes. In addition, although dopaminergic and glutamatergic influences in the NAc have been implicated in instrumental learning, it is unclear whether similar mechanisms operate during Pavlovian conditioning. To evaluate these issues, the effects of posttraining intra-NAc infusions of D1, D2, and NMDA receptor antagonists, as well as d-amphetamine, were determined on Pavlovian autoshaping in rats, which assesses learning by discriminated approach behavior to a visual conditioned stimulus predictive of food reward. Intracerebral infusions were given either immediately after each conditioning session to disrupt early memory consolidation or after a delay of 24 h. Findings indicate that immediate, but not delayed, infusions of both D1 (SCH 23390) and NMDA (AP-5) receptor antagonists significantly impair learning on this task. By contrast, amphetamine and the D2 receptor antagonist sulpiride were without significant effect. These findings provide the most direct demonstration to date that D1 and NMDA receptors in the NAc contribute to, and are necessary for, the early consolidation of appetitive Pavlovian learning (Dalley, 2005; full text of article).

Dopamine receptor and goal-directed behavior

Intracranial self-stimulation (ICSS) activates the neural pathways that mediate reward, including dopaminergic terminal areas such as the nucleus accumbens (NAc). However, a direct role of dopamine in ICSS-mediated reward has been questioned. Simultaneous voltammetric and electrophysiological recordings from the same electrode reveal that, at certain sites, the onset of anticipatory dopamine surges and changes in neuronal firing patterns during ICSS are coincident, whereas sites lacking dopamine changes also lack patterned firing. Intrashell microinfusion of a D1, but not a D2 receptor antagonist, blocks ICSS. An iontophoresis approach was implemented to explore the effect of dopamine antagonists on firing patterns without altering behavior. Similar to the microinfusion experiments, ICSS-related firing is selectively attenuated following D1 receptor blockade. This work establishes a temporal link between anticipatory rises of dopamine and firing patterns in the NAc shell during ICSS and suggests that they may play a similar role with natural rewards and during drug self-administration (Cheer, 2007).

Dopamine receptor hippocampal synaptic plasticity

Hebbian learning models require that neurons are able to both strengthen and weaken their synaptic connections. Hippocampal synaptic plasticity, in the form of long-term potentiation (LTP) and long-term depression (LTD), has been implicated in both spatial memory formation as well as novelty acquisition. In addition, the ventral tegmental area-hippocampal loop has been proposed to control the entry of information into long-term memory, whereas the dopaminergic system is believed to play an important role in information acquisition and synaptic plasticity. D1/D5 dopamine receptors are positively coupled to adenylyl cyclase and have been to modulate certain forms of synaptic plasticity, particularly in vitro. This study investigated how D1/D5 dopamine receptors modify long-lasting synaptic plasticity at CA1 synapses of adult freely moving rats and found that receptor activation lowered the threshold for the induction of both LTP and LTD. Specific types of learning are associated with specific types of hippocampal synaptic plasticity. Object-configuration learning, facilitation of late-phase LTD by object exploration, and late-phase LTP by exploration of empty space were all prevented by D1/D5 receptor antagonism. Furthermore, receptor antagonism prevented electrically induced late-LTP, whereas receptor activation facilitated induction of both LTP and LTD by patterned electrical stimulation. These findings suggest that the dopaminergic system, acting via D1/D5 receptors, gates long-term changes in synaptic strength and that these changes are a critical factor in the acquisition of novel information (Lemon, 2006: full text of article).


Search PubMed for articles about Drosophila Dopamine 1-like receptor 1

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Handler, A., Graham, T. G. W., Cohn, R., Morantte, I., Siliciano, A. F., Zeng, J., Li, Y. and Ruta, V. (2019). Distinct dopamine receptor pathways underlie the temporal sensitivity of associative learning. Cell 178(1): 60-75. PubMed ID: 31230716

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Biological Overview

date revised: 12 December 2022

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