The Interactive Fly

Zygotically transcribed genes

Genes Regulating Synthesis and Function of Biogenic Amines

Neuromodulation: Dopaminergic, Octopaminergic and Serotonergic Nervous Systems



Biogenic Amines, Learning and Memory
  • tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals
  • Neuroarchitecture of aminergic systems in the larval ventral ganglion of Drosophila melanogaster
  • Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila
  • Activity of defined mushroom body output neurons underlies learned olfactory behavior in Drosophila
  • Dopaminergic neurons write and update memories with cell-type-specific rules
  • Circuit analysis of a Drosophila Dopamine type 2 receptor that supports anesthesia-resistant memory
  • Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster
  • Pharmacological characterization of the fifth serotonin receptor subtype of Drosophila melanogaster
  • Dopamine neurons modulate pheromone responses in Drosophila courtship learning
  • Heterosynaptic plasticity underlies aversive olfactory learning in Drosophila
  • Coordinated and compartmentalized neuromodulation shapes sensory processing in Drosophila
  • Trace conditioning in Drosophila induces associative plasticity in mushroom body kenyon cells and dopaminergic neurons
  • Re-evaluation of learned information in Drosophila
  • Coincident postsynaptic activity gates presynaptic dopamine release to induce plasticity in Drosophila mushroom bodies
  • Pre- and postsynaptic role of Dopamine D2 Receptor DD2R in Drosophila olfactory associative learning
  • Suppression of dopamine neurons mediates reward
  • Novel role for ecdysone in Drosophila conditioned behavior: Linking GPCR-mediated non-canonical steroid action to cAMP signaling in the adult brain
  • Autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling
  • Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling
  • Layered reward signalling through octopamine and dopamine in Drosophila
  • Flight initiation and maintenance deficits in flies with genetically altered biogenic amine levels
  • Isoform- and cell-specific function of tyrosine decarboxylase in the Drosophila Malpighian tubule
  • Octopamine mediates starvation-induced hyperactivity in adult Drosophila
  • Octopamine controls starvation resistance, life span and metabolic traits in Drosophila
  • Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila
  • PPL2ab neurons restore sexual responses in aged Drosophila males through dopamine
  • Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain
  • Electrochemical measurements of optogenetically stimulated quantal amine release from single nerve cell varicosities in Drosophila larvae
  • Four individually identified paired dopamine neurons signal reward in larval Drosophila
  • Disruption of dopamine homeostasis has sexually dimorphic effects on senescence characteristics of Drosophila melanogaster
  • Active and passive sexual roles that arise in Drosophila male-male courtship are modulated by dopamine levels in PPL2ab neurons
  • A decision underlies phototaxis in an insect]
  • Reciprocal synapses between mushroom body and dopamine neurons form a positive feedback loop required for learning

    Biogenic amines and Aggression
  • Octopamine in male aggression of Drosophila
  • A subset of octopaminergic neurons are important for Drosophila aggression
  • Single serotonergic neurons that modulate aggression in Drosophila
  • Modulation of Drosophila male behavioral choice
  • Drosophila seminal protein Ovulin mediates ovulation through female octopamine neuronal signaling
  • Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster

    Biogenic Amines, Appetite Control and Food Choice
  • Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing
  • Octopamine-mediated circuit mechanism underlying controlled appetite for palatable food in Drosophila
  • Dopaminergic modulation of sucrose acceptance behavior in Drosophila
  • A higher brain circuit for immediate integration of conflicting sensory information in Drosophila
  • Serotonergic modulation enables pathway-specific plasticity in a developing sensory circuit in Drosophila
  • A subset of serotonergic neurons evokes hunger in adult Drosophila
  • Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger
  • A single pair of serotonergic neurons counteracts serotonergic inhibition of ethanol attraction in Drosophila
  • Dynamics of memory-guided choice behavior in Drosophila
  • Coordinated and compartmentalized neuromodulation shapes sensory processing in Drosophila
  • Neural circuits for long-term water-reward memory processing in thirsty Drosophila
  • Serotonergic network in the subesophageal zone modulates the motor pattern for food intake in Drosophila

    Biogenic Amines and Sleep
  • Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits
  • Identification of a dopamine pathway that regulates sleep and arousal in Drosophila
  • Caffeine promotes wakefulness via dopamine signaling in Drosophila
  • Identified serotonin-releasing neurons induce behavioral quiescence and suppress mating in Drosophila
  • Drosophila D1 dopamine receptor mediates caffeine-induced arousal
  • D1 receptor activation in the mushroom bodies rescues sleep-loss-induced learning impairments in Drosophila
  • Identification of a neural circuit that underlies the effects of octopamine on sleep:wake behavior
  • Octopamine regulates sleep in Drosophila through protein kinase A-dependent mechanisms
  • Sleep facilitates memory by blocking dopamine neuron-mediated forgetting
  • Control of sleep by dopaminergic inputs to the Drosophila mushroom body
  • Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain
  • Characterization of a novel Drosophila SERT mutant: insights on the contribution of the serotonin neural system to behaviors
  • The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for MDMA translocation and the induction of substrate efflux
  • Modulatory action by the serotonergic system: behavior and neurophysiology in Drosophila melanogaster
  • Visual attention in flies-dopamine in the mushroom bodies mediates the after-effect of cueing
  • Operation of a homeostatic sleep switch
  • Serotonin modulates a depression-like state in Drosophila responsive to lithium treatment
  • Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila

    Biogenic Amines and Development
  • Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis
  • Limited distal organelles and synaptic function in extensive monoaminergic innervation
  • Genes involved in synthesis of biogenic amines

  • Genes regulating the synthesis and function of biogenic amines


    tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals

    In Drosophila melanogaster, ebony and tan, two cuticle melanizing mutants, regulate the conjugation (ebony) of β-alanine to dopamine or hydrolysis (tan) of the β-alanyl conjugate to liberate dopamine. β-alanine biosynthesis is regulated by black. ebony and tan also exert unexplained reciprocal defects in the electroretinogram, at ON and OFF transients attributable to impaired transmission at photoreceptor synapses, which liberate histamine. Compatible with this impairment, both mutants have reduced histamine contents in the head, as measured by HPLC, and have correspondingly reduced numbers of synaptic vesicles in their photoreceptor terminals. Thus, the histamine phenotype is associated with sites of synaptic transmission at photoreceptors. When they receive microinjections into the head, wild-type Sarcophaga bullata (in whose larger head such injections are routinely possible) rapidly (<5 sec) convert exogenous [3H]histamine into its β-alanine conjugate, carcinine, a novel metabolite. Drosophila tan has an increased quantity of [3H]carcinine, the hydrolysis of which is blocked; ebony lacks [3H]carcinine, which it cannot synthesize. Confirming these actions, carcinine rescues the histamine phenotype of ebony, whereas β-alanine rescues the carcinine phenotype of black;tan double mutants. The equilibrium ratio between [3H]carcinine and [3H]histamine after microinjecting wild-type Sarcophaga favors carcinine hydrolysis, increasing to only 0.5 after 30 min. These findings help resolve a longstanding conundrum of the involvement of tan and ebony in photoreceptor function. It is suggested that reversible synthesis of carcinine occurs in surrounding glia, serving to trap histamine after its release at photoreceptor synapses; subsequent hydrolysis liberates histamine for reuptake (Borycz, 2002).

    Activity of defined mushroom body output neurons underlies learned olfactory behavior in Drosophila

    During olfactory learning in fruit flies, dopaminergic neurons assign value to odor representations in the mushroom body Kenyon cells. This study identified a class of downstream glutamatergic mushroom body output neurons (MBONs) called M4/6, or MBON-β2β'2a, MBON-β'2mp, and MBON-γ5β'2a, whose dendritic fields overlap with dopaminergic neuron projections in the tips of the β, β', and γ lobes. This anatomy and their odor tuning suggests that M4/6 neurons pool odor-driven Kenyon cell synaptic outputs. Like that of mushroom body neurons, M4/6 output is required for expression of appetitive and aversive memory performance. Moreover, appetitive and aversive olfactory conditioning bidirectionally alters the relative odor-drive of M4β' neurons (MBON-β'2mp). Direct block of M4/6 neurons in naive flies mimics appetitive conditioning, being sufficient to convert odor-driven avoidance into apprroach, while optogenetically activating these neurons induces avoidance behavior. It is therefore proposed that drive to the M4/6 neurons reflects odor-directed behavioral choice. See Three Pairs of Glutamatergic Output Neurons Innervate the Tips of the Horizontal Mushroom Body Lobes (Owald, 2015).

    Many prior studies have concluded that mushroom body neurons are dispensable for naive odor-driven behavior and subsets are either required or are dispensable for particular memory functions. However, these experiments simultaneously blocked all the outputs from a given population of KCs using cell-wide expression of shits1. The current results suggest that these models should be reconsidered. Blocking the specific M4β/MBON-β2β'2a, M4β'/MBON-β'2mp, and M6/MBON-γ5β'2a output from the mushroom body, as opposed to blocking all outputs, has a radical effect on naive odor-driven behavior. It is proposed that ordinarily, in naive flies, the multiple mushroom body output channels are ultimately pooled and contribute a net zero to odor-driven behavior. Therefore, if one uses a mushroom body neuron-driven UAS-shits1 that simultaneously blocks all outputs, there is no apparent effect on naive behavior. If, however, one blocks only one channel, or alters its efficacy by learning, the odor-driven behavior can be changed. A similar logic could also account for why clear memory retrieval defects are seen when blocking M4β'/MBON-β'2mp and M6/MBON-γ5β'2a neurons that presumably pool outputs from the tip of the γ and β' lobe, yet blocking all α'β' neuron outputs did not demonstrably disrupt later memory retrieval. Others have shown a role for α'β' neuron output to retrieve earlier forms of memory (Owald, 2015).

    Both the physiological and behavioral results are consistent with a depression of the M4β'/MBON-β'2mp and M6/MBON-γ5β'2a output being sufficient to code learned approach. Learning-related plasticity has been reported at the β-lobe outputs in both bees and locusts, although the importance of these synaptic connections in the behavior of these insects is not known. At this stage it is not certain that the observed decrease in the relative odor drive reflects plasticity of the synapses between odor-specific KCs and the M4/6 neurons. However, it seems plausible, because this synaptic junction is addressed by the relevant rewarding dopaminergic neurons. Given that blocking M4β'/MBON-β'2mp and M6/MBON-γ5β'2a neurons converts avoidance to approach, other mushroom body output channels, perhaps some of which lie on the vertical α-lobe projection, must drive the approach behavior. It is therefore conceivable that a similar plasticity of odor drive to these putative approach outputs could be critical for aversive conditioning. Such an idea is consistent with several prior reports of aversive memory traces that are specific to the vertical α-branch of the mushroom body. In addition, aversive learning has been reported to depress odor drive in the vertical lobe of downstream MB-V2α/MBON-α2sc and MB-V2α'/MBON-α'3 neurons and to potentiate odor drive of MB-V3/MBON-α3 output neurons. However, it is notable that blocking either the MB-V2α/MBON-α2sc and MB-V2α'/MBON-α'3 neurons or MB-V3/MBON-α3 neurons did not affect naive odor avoidance behavior in the current experiments or those of others. Therefore, although MB-V2α/MBON-α2sc, MB-V2α'/MBON-α'3, and MB-V3/MBON-α3 neurons are required for memory expression, it is not currently known which reinforcing neurons address MB-V2α/MBON-α2sc, MB-V2α'/MBON-α'3, and MB-V3/MBON-α3 connections and how these outputs specifically contribute to odor-guided behavior (Owald, 2015).

    The physiological analyses suggest bidirectional plasticity of odor-evoked responses, with aversive learning increasing the relative conditioned odor drive to the M4β'/MBON-β'2mp neurons. This could account for why output from M4/6 neurons is also required for expression of aversive memory. Moreover, whereas blocking the M4β/MBON-β2β'2a, M4β'/MBON-β'2mp, and M6/MBON-γ5β'2a neurons converts odor avoidance into approach, activation of M4β/MBON-β2β'2a, M4β'/MBON-β'2mp, and M6/MBON-γ5β'2a neurons drives avoidance. It therefore seems likely that plasticity of the relative odor drive to M4β'/MBON-β'2mp neurons is also part of the aversive memory engram. Again, it is not known that the increased odor drive after training reflects synaptic potentiation between odor-specific KCs and the M4β'/MBON-β'2mp neurons. Increased odor drive to M4β'/MBON-β'2mp neurons could, for example, also result from plasticity elsewhere in the KCs that enhances signal propagation along the horizontal KC arbor. Nevertheless, the MB-M3 dopaminergic neurons that are required to reinforce aversive memory also innervate the tips of the β and β' lobe. In addition, a recent study reported that aversive learning specifically decreased unconditioned odor-evoked neurotransmission from the γ neurons, a result that presumably would mirror a relative increase in the response to the reinforced odor. Lastly, aversive conditioning using relative shock intensity utilizes the rewarding dopaminergic neurons that occupy the same zones on the mushroom body as the M4β'/MBON-β'2mp and M6/MBON-γ5β'2a neuron dendrites. With the caveat that GRASP is only an indicator of proximity, the anatomical studies suggest that dendrites of rewarding dopaminergic neurons may connect to the M4β'/MBON-β'2mp and M6/MBON-γ5β'2a neuron presynaptic terminals, forming a potential feedback or forward loop that could serve such a relative-judgment function (Owald, 2015).

    It is perhaps noteworthy that KC outputs in the vertical lobe are onto excitatory cholinergic MB-V2α/MBON-α2sc and MB-V2α'/MBON-α'3 neurons, whereas the horizontal outputs are onto glutamatergic, potentially inhibitory, M4β/MBON-β2β'2a, M4β'/MBON-β'2mp, and M6/MBON-γ5β'2a neurons. This suggests that distinct signaling modes may be driven from the bifurcated collaterals of KCs. It will be crucial to understand how these outputs from the different branches, and those from discrete lobes, are ultimately pooled to guide appropriate behavior (Owald, 2015).

    Dopaminergic neurons write and update memories with cell-type-specific rules

    Associative learning is thought to involve parallel and distributed mechanisms of memory formation and storage. In Drosophila, the mushroom body (MB) is the major site of associative odor memory formation. The anatomy of the adult MB and 20 types of dopaminergic neurons (DANs) that each innervate distinct MB compartments have been previously defined and described. This study compared the properties of memories formed by optogenetic activation of individual DAN cell types. Extensive differences were found in training requirements for memory formation, decay dynamics, storage capacity and flexibility to learn new associations. Even a single DAN cell type can either write or reduce an aversive memory, or write an appetitive memory, depending on when it is activated relative to odor delivery. These results show that different learning rules are executed in seemingly parallel memory systems, providing multiple distinct circuit-based strategies to predict future events from past experiences. The mechanisms that generate these distinct learning rules are unknown. They could arise from differences in the dopamine release properties of different DAN cell types or from local differences in the biochemical response to dopamine signaling in each MB compartment. For example, KCs express four distinct dopamine receptors, which might be deployed differently in each compartment. Or they could originate from circuit properties (Aso, 2016).

    Circuit analysis of a Drosophila Dopamine type 2 receptor that supports anesthesia-resistant memory

    Dopamine is central to reinforcement processing and exerts this function in species ranging from humans to fruit flies. It can do so via two different types of receptors (i.e., D1 or D2) that mediate either augmentation or abatement of cellular cAMP levels. Whereas D1 receptors are known to contribute to Drosophila aversive odor learning per se, this study shows that D2 receptors are specific for support of a consolidated form of odor memory known as anesthesia-resistant memory. By means of genetic mosaicism, this function was localized to Kenyon cells, the mushroom body intrinsic neurons, as well as GABAergic APL neurons and local interneurons of the antennal lobes, suggesting that consolidated anesthesia-resistant memory requires widespread dopaminergic modulation within the olfactory circuit. Additionally, dopaminergic neurons themselves require D2R, suggesting a critical role in dopamine release via its recognized autoreceptor function. Considering the dual role of dopamine in balancing memory acquisition (proactive function of dopamine) and its 'forgetting' (retroactive function of dopamine), this analysis suggests D2R as central player of either process (Scholz-Kornehl, 2016).

    Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster

    Neuromodulation confers flexibility to anatomically-restricted neural networks so that animals are able to properly respond to complex internal and external demands. However, determining the mechanisms underlying neuromodulation is challenging without knowledge of the functional class and spatial organization of neurons that express individual neuromodulatory receptors. This study describes the number and functional identities of neurons in the antennal lobe of Drosophila melanogaster that express each of the receptors for one such neuromodulator, serotonin (5-HT). Although 5-HT enhances odor-evoked responses of antennal lobe projection neurons (PNs) and local interneurons (LNs), the receptor basis for this enhancement is unknown. Endogenous reporters of transcription and translation for each of the five 5-HT receptors (5-HTRs) were used to identify neurons, based on cell class and transmitter content, that express each receptor. Specific receptor types are expressed by distinct combinations of functional neuronal classes. For instance, the excitatory PNs express the excitatory 5-HTRs (5-HT2 type and 5-HT7), the 5-HT1 type receptors are generally inhibitory, and distinct classes of LNs each express different 5-HTRs. This study therefore provides a detailed atlas of 5-HT receptor expression within a well-characterized neural network, and enables future dissection of the role of serotonergic modulation of olfactory processing (Sizemore, 2016).

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

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

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

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

    Pharmacological characterization of the fifth serotonin receptor subtype of Drosophila melanogaster

    Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster, a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT1A, Dm5-HT1B, and Dm5-HT7 couple to cAMP signaling cascades, the Dm5-HT2A receptor leads to Ca2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT2B receptor. Knowledge about this receptor's pharmacological properties is very limited. This is quite surprising because Dm5-HT2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT2B's pharmacology, this study evaluated the receptor's response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT2B signaling in vitro and in vivo (Blenau, 2017).

    Coordinated and compartmentalized neuromodulation shapes sensory processing in Drosophila

    Learned and adaptive behaviors rely on neural circuits that flexibly couple the same sensory input to alternative output pathways. This study shows that the Drosophila mushroom body functions like a switchboard in which neuromodulation reroutes the same odor signal to different behavioral circuits, depending on the state and experience of the fly (see Compartmentalized Architecture of the Mushroom Body). Using functional synaptic imaging and electrophysiology, it was shown that dopaminergic inputs to the mushroom body modulate synaptic transmission with exquisite spatial specificity, allowing individual neurons to differentially convey olfactory signals to each of their postsynaptic targets. Moreover, the dopaminergic neurons function as an interconnected network, encoding information about both an animal's external context and internal state to coordinate synaptic plasticity throughout the mushroom body. These data suggest a general circuit mechanism for behavioral flexibility in which neuromodulatory networks act with synaptic precision to transform a single sensory input into different patterns of output activity (Cohn, 2015).

    This study took advantage of the mushroom body's orderly architecture to gain insight into the circuit mechanisms through which neuromodulation mediates flexible sensory processing. Compartmentalized dopaminergic signaling permits independent tuning of synaptic transmission between an individual KC and its repertoire of postsynaptic MBON targets. As a consequence, the same KC odor representation can evoke different patterns of output activity, depending on the state of the animal and the dopaminergic network. Recent data indicate that the ensemble of MBONs acts in concert to bias an animal's behavioral response to an odor such that altering the balance of their activity can modify the olfactory preferences of both naive and trained animals. In accord with such a model, this study revealed how a distributed neuromodulatory network is poised to orchestrate plasticity across all 15 compartments of the mushroom body and reweight the net output of the MBONs, allowing for adaptive behavioral responses based on the immediate needs or past experience of the animal (Cohn, 2015).

    Distinct subsets of DANs are sufficient to drive learned olfactory associations, leading to the suggestion they may act autonomously to encode the rewarding or punishing contextual stimuli that assign meaning to an odor. The current data, however, suggest a more complex circuit architecture, in which rich functional interconnectivity between compartments contributes to coordinated and bidirectional patterns of activity across the DAN population. This raises the possibility that reinforcement experiences may be represented by combinatorial patterns of DAN excitation and inhibition in different compartments, endowing the dopaminergic population with a greater capacity to instruct behavior via the limited repertoire of mushroom body outputs. Intriguingly, midbrain dopaminergic neurons responsive to punishment and reward also project to distinct targets in the mammalian brain and display a similar functional opponency as a consequence of reciprocal network interactions. Thus, the concerted and partially antagonistic action of neuromodulatory pathways may represent a general and conserved circuit principle for generating adaptive behavioral responses (Cohn, 2015).

    Distinct DAN network activity states are evoked by electric shock and sugar ingestion, reinforcers classically used in associative olfactory conditioning paradigms because of their strong inherent valence. However, similarly distributed patterns of DAN activity are correlated with the fly's motor activity, implying that an animal's behavioral state might serve as a reinforcement stimulus that itself drives synaptic plasticity to shape odor processing. Metabolic states, such as thirst and hunger, have been shown to gate appetitive reinforcement by water and sugar rewards, permitting state-dependent formation of olfactory associations only in motivated animals. The current data highlight an additional facet of how an animal's internal state can regulate dopamine release to adjust the salience of contextual cues. Together, these observations indicate that the distributed DAN network integrates information about external context and internal state with MBON feedback to represent the moment-by-moment experience of an animal and dynamically regulate the flow of olfactory signals through the mushroom body (Cohn, 2015).

    The independent regulation of synapses along an axon is thought to permit a single neuron to convey specialized information to different downstream targets, providing additional flexibility and computational power to neural circuits. In the mushroom body, synapse-specific plasticity is achieved through spatially restricted patterns of dopaminergic modulation that divide a KC axon into functionally distinct segments. Thus, the ensemble of synapses within a compartment, as the site of convergence for sensory and contextual signals, represents the elementary functional unit that underlies experience-dependent mushroom body output (Cohn, 2015).

    Within a compartment, multiple neuromodulatory mechanisms appear to shape synaptic signaling. Broad potentiation of KC-MBON synapses is seen after DAN activation, but odor-specific depression is seen if DANs were coincidently activated with KCs, consistent with the synaptic changes previously proposed to occur after learning. Taken together, these findings indicate that neuromodulation in the mushroom body instructs opposing forms of synaptic plasticity, analogous to the bidirectional tuning of synaptic strength by dopamine in mammalian brain centers. The molecular mechanisms through which dopamine can direct diverse synaptic changes within a compartment remain to be elucidated, but they may depend on signaling through different dopamine receptors or downstream signaling cascades that function as coincidence detectors. Indeed, while DopR1 in KCs is essential to the formation of learned olfactory associations, this receptor was found to play only a subtle role in the context-dependent patterning of Ca2+ along their axons. Conversely, DopR2 strongly influences the topography of presynaptic Ca2+ along KC axons, in accord with evidence that tonic release of dopamine during ongoing behavior acts through this receptor to interfere with the maintenance of specific learned olfactory associations. Thus, distinct molecular pathways may transform the same dopaminergic reinforcement signals into synaptic changes of opposite polarity to shape olfactory processing based on both the present context and prior experiences of an individual (Cohn, 2015).

    The mushroom body has been most extensively studied as a site for associative learning in which the temporal pairing of an odor with a reinforcement experience selectively alters subsequent behavioral responses to that odor. The current data suggest that the convergence of DAN network activity and KC olfactory representations within the mushroom body lobes may drive associative plasticity in each compartment, allowing the odor tuning of the MBON repertoire to reflect the unique experiences of an individual. However, these observations also provide insight into the mushroom body's broader role in the context-dependent regulation of innate behaviors. The ongoing activity of the distributed DAN network, encoding information about an animal's current environmental context and behavioral state, is poised to continuously reconfigure the activity patterns of the MBON population to allow for adaptive responses based on the acute needs of the animal. This context-dependent synaptic modulation could potentially erode odor-specific learned associations within the mushroom body, permitting the immediate circumstances of an animal to dominate over previously learned olfactory associations that may no longer be predictive or relevant. The axons of MBONs ultimately converge with output pathways from the lateral horn, a Drosophila brain center thought to mediate stereotyped responses to odors, providing a potential substrate for learned and context-dependent output from the mushroom body to influence inherent olfactory preferences (Cohn, 2015).

    Thus, the dual role of neuromodulation in the mushroom body-to select among alternative circuit states that regulate both innate and learned behaviors-is reminiscent of its function in other higher integrative brain centers. In the basal ganglia, for example, different temporal patterns of dopamine release are thought to select the relevant circuit configurations that control inherently motivated behaviors as well as reinforcement learning. The generation of flexible behavioral responses based on experience, whether past or present, may therefore rely on common integrative brain structures in which neuromodulatory networks act with exquisite spatial precision to shape sensory processing (Cohn, 2015).

    Trace conditioning in Drosophila induces associative plasticity in mushroom body kenyon cells and dopaminergic neurons

    Dopaminergic neurons (DANs) signal punishment and reward during associative learning. In mammals, DANs show associative plasticity that correlates with the discrepancy between predicted and actual reinforcement (prediction error) during classical conditioning. Also in insects, such as Drosophila, DANs show associative plasticity that is, however, less understood. This study examined ssociative plasticity in DANs and their synaptic partners, the Kenyon cells (KCs) in the mushroom bodies (MBs), while training Drosophila to associate an odorant with a temporally separated electric shock (trace conditioning). In most MB compartments DANs strengthened their responses to the conditioned odorant relative to untrained animals. This response plasticity preserved the initial degree of similarity between the odorant- and the shock-induced spatial response patterns, which decreased in untrained animals. Contrary to DANs, KCs (α'/β'-type) decreased their responses to the conditioned odorant relative to untrained animals. No evidence was found for prediction error coding by DANs during conditioning. Rather, the data supports the hypothesis that DAN plasticity encodes conditioning-induced changes in the odorant's predictive power (Dylla, 2017).

    Associative learning enables animals to anticipate negative or positive events. The neural mechanisms of associative learning are commonly studied in classical conditioning paradigms, in which animals are trained to associate a cue (conditioned stimulus; CS) with a punishment or reward (unconditioned stimulus; US). In the standard conditioning paradigm CS and US overlap in time, while in the trace conditioning paradigm there is a temporal gap between the CS and US. During both standard conditioning and trace conditioning, the US is mediated by dopaminergic neurons (DANs), in animals as diverse as monkeys and fruit flies (Dylla, 2017).

    Genetic tools for monitoring and manipulating neuronal activity in the fruit fly Drosophila melanogaster promoted the understanding of the neural mechanisms of dopamine-mediated learning. Those mechanisms are well-described for standard 'odor-shock conditioning' in Drosophila, in which an olfactory CS is paired with a temporally overlapping electric shock US. During conditioning, an odor-shock association is formed in the mushroom body (MB) neuropil. The intrinsic neurons of the MB, the Kenyon cells (KCs), receive olfactory input in the MB-calyx and project to the vertical (α and α'), and the medial (β, β', and γ) MB-lobes. During odor-shock conditioning, the olfactory CS activates an odorant-specific KC population, and the electric shock US activates DANs that innervate the MB-lobes. In KCs, the CS-induced increase in intracellular calcium and the US-(dopamine)-induced second messengers synergistically activate an adenylyl cyclase, which alters the synaptic strength between KCs and MB output neurons (MBONs). This change in KC-to-MBON synapses is thought to encode the associative odor memory (Dylla, 2017).

    The MB-lobes are divided into 15 compartments (α1-3, β1-2, α'1-3, β'1-2, and γ1-5), each of which is innervated by a distinct population of DANs and MBONs. These compartments constitute functional units, which are involved in different forms of associative learning. In compartments such as γ1, γ2, and β2, DANs mediate electric shock reinforcement. Besides mediating reinforcement during classical conditioning, Drosophila DANs are involved in long-term memory formation, forgetting, extinction learning and memory reconsolidation, and in integrating internal states with memory and sensory processing. A single DAN can even serve different functions, for example, PPL1-γ1pedc (also referred to as MB-MP1) signals reinforcement, and controls state-dependent memory retrieval (Dylla, 2017).

    The functional complexity of Drosophila DANs is further increased by the fact that DANs show learning-induced associative plasticity: they increase their response to the CS during classical conditioning. Mammalian DANs also increase their CS-induced responses during classical conditioning. In addition, they decrease their response to the US, and when a predicted US does not occur, they decrease their activity below baseline level. This pattern of response plasticity in mammalian DANs is compatible with the hypothesis that animals only learn to associate a CS with a US, when the US occurs unpredictably. Thus, mammalian DANs appear to encode this prediction error. In Drosophila, however, DANs do not change their response to the US. Therefore, Drosophila DANs appear to encode the US prediction by the CS rather than encoding the US prediction error during classical conditioning. It is not clear, whether classical conditioning in insects is driven by US prediction error. There is evidence for prediction error-driven conditioning in crickets, but there is also a controversy about whether or not blocking (a failure to learn, when the US is already predicted by another CS) occurs (Dylla, 2017).

    This study reassessed the hypothesis that Drosophila DANs do not encode the prediction error during classical conditioning (Riemensperger, 2005). Different from Riemensperger (2005) who pooled DAN activity across the mushroom body lobes, this study differentiated between DAN types that innervate different compartments of the MB lobes. Moreover, instead of using standard conditioning, trace conditioning with a 5 s gap between the CS and the US was used, allowing for precise distinguishing between responses to either the CS or the US (Dylla, 2017).

    This study investigated associative plasticity in the responses of DANs and their synaptic partners, the KCs, across the compartments of the Drosophila MB. Using calcium imaging, CS- and US-induced responses of a subpopulation of DANs (labeled by TH-GAL4) and of KCs (labeled by OK107-GAL4) were recorded during odor-shock trace conditioning. Note, that most compartments are innervated by multiple TH-GAL4-labeled DANs. Therefore, the average activity that was recorded in most of the compartments might mask possible differences in the response properties and plasticity between individual DANs and KCs. Only DAN responses in the compartments γ2 and α'1 reflect the responses of a single neuron (Dylla, 2017).

    Across MB compartments, DANs and KCs differed in their response strength to odorants and electric shock, and they differed in CS-US pairing-induced plasticity. Compared to the unpaired control groups, KCs decreased their responses to the CS in all compartments of the β'-lobe and in the junction, while DANs increased their responses to the CS in all compartments of the γ- and β'-lobe, and in the junction. The occurrence of associative plasticity in DANs in the compartments γ3-5 and β'1 is surprising, given that these DANs are not known to be involved in odor-shock conditioning, after training there was neither an associative change in US-induced DAN responses nor a change of activity during US-omission after CS presentation. It is therefore concluded that Drosophila DANs do not encode the US-prediction error during classical conditioning (Dylla, 2017).

    Previous studies suggested that DANs in the MB lobes respond strongly to electric shock and weakly to odorants. The compartment-resolved analysis of the calcium imaging data refines this picture: It is confirmed that DANs of all imaged compartments respond to both electric shock and odorants, and it was shown that their relative response strength to odorants and electric shock differs across compartments. For example, DANs innervating γ1 responded stronger to electric shock than to odorants, while DANs innervating β'2 responded equally strong to odorants and electric shock. The strongest DAN responses to electric shock were shown to be in the compartments γ1 and γ2. These compartments receive input from PPL1-γ1pedc and PPL1-γ2α'1 DANs that mediate electric shock reinforcement. In all compartments, except in α1/α'1, the DAN response strength correlated positively with the current strength encountered by individual flies. Thus, DANs are capable of encoding the strength of the electric shock US, and this property may account for the positive dependence between electric shock strength and learning performance in flies (Dylla, 2017).

    Calcium responses in KCs differ between MB lobes, and they differ between the compartments of a given lobe, possibly due to compartment-specific modulation by DANs and MBONs. KCs in γ2 and γ3 responded strongest to odorants, confirming the results of Cohn (2015). KCs generally responded only weakly to electric shocks. Previously published strong KC responses to electric shock may be because electric shocks were applied to the flies' abdomen rather than to their legs, which might have resulted in a stronger stimulation (Dylla, 2017).

    The associative strengthening of DAN responses to the olfactory CS (as compared to the unpaired control group), confirms the previous report by Riemensperger (2005). Associative plasticity occurred in those DANs that innervate the MBs (PPL1 and PAM cluster DANs; note that the used TH-GAL4 driver line covers only a small subpopulation of PAM neurons but not in DANs that innervate the central complex (PPL1 and PPM3 cluster DANs). This is in line with the established role of MB innervating-DANs in associative memory formation, while central complex-innervating DANs are involved in behaviors such as locomotion, wakefulness, arousal, and aggression, and are therefore not expected to show odor-shock conditioning-induced plasticity (Dylla, 2017).

    In contrast to previous studies, this study did not find an associative increase in KC calcium responses to the CS in the MB-lobes after odor-shock conditioning. This may indicate either a difference between trace conditioning and standard conditioning, or a difference in other experimental parameters that may also account for inconsistencies in the published effects of odor-shock conditioning (Dylla, 2017).

    The associative decrease in KC responses in the β'-lobe compartments is in line with previous studies that showed conditioning-induced depression of KC-to-MBON synapses (Cohn, 2015; Hige, 2015). Therefore, it propose that the associative decrease in KC responses to the CS reflects a presynaptic depression at KC-to-MBON synapses in β'-lobe compartments (Dylla, 2017).

    What is the site of neuronal plasticity that underlies the relative increase in DANs' responses to the olfactory CS? Riemensperger (2005) proposed that DANs get odorant-driven excitatory input via a MBON feedback loop that is strengthened during odor-shock conditioning. However, the DAN population is composed of different neuron types that do not share a common input either from MBONs or from other neurons that could explain the global associative plasticity across MB compartments. Because KCs presumably provide the only common odor-driven input to all MB-innervating DANs, it is suggested that the site of associative plasticity is located in a KC-to-DAN synapse. Indeed, KC-to-DAN synapses have recently been reported in Drosophila (Cervantes-Sandoval, 2017). Associative increase in CS-induced DAN responses occurred despite unaltered or decreased KC responses in the same compartment. This suggests that the associative plasticity occurs post-synaptic in DANs and is not inherited from KCs (Dylla, 2017).

    What is the neuronal substrate of CS-US coincidence detection in DANs and KCs? Drosophila trace conditioning depends on dopamine receptor-triggered signaling in KCs, as is the case for standard conditioning. However, the CS-US coincidence detection mechanism in trace conditioning is unknown. In standard conditioning the CS-induced increase in KCs' calcium concentration coincides with the US-(dopamine)-induced second messengers, which is thought to synergistically activate the rutabaga adenylyl cyclase, and ultimately alters the strength of KC-to-MBON synapses. This mechanism would not work for trace conditioning, because (1) at the time the US occurs, CS-induced increase in KCs' calcium concentration is back to baseline levels, and (2) trace conditioning does not involve the rutabaga adenylyl cyclase. It is therefore hypothesized that a non-rutabaga adenylyl cyclase or a protein kinase C could serve as a molecular coincidence detector for the CS trace and the US. For example, the CS-induced calcium and dopamine signaling could lead to a sustained activation of an adenylyl cyclase or protein kinase C in KCs, which then would increase synergistically and drive synaptic plasticity during the US-induced dopamine signaling (Dylla, 2017).

    DAN responses to odorants and associative strengthening of DAN responses to the CS-odorant are not included in current models of associative learning in the MB. However, associative plasticity is a common feature of US-mediating neurons, which occurs in mammalian and Drosophila DANs, and in an octopaminergic neuron in honey bees (Dylla, 2017).

    What could be the function of odorant-induced responses and odor-shock conditioning-induced plasticity in DANs? MB-innervating DANs strengthened their response to the CS (as compared to the unpaired group) during odor-shock conditioning, in line with Riemensperger (2005). However, other than in monkey DANs, this study did not observe associative plasticity in DANs' response to the US. The data therefore support the idea that Drosophila DANs encode predictive power of the CS, e.g., US-prediction, but not the US-prediction error during classical conditioning (Dylla, 2017).

    This study found shock-induced responses and associative plasticity in DANs that are not involved in odor-shock conditioning, for example in DANs innervating β'1, γ3, γ4, and γ5. This suggests that those DANs serve a function in aversive odor learning which is not captured by the commonly applied conditioning paradigms. For example, the relative strengthening of CS-induced responses could mediate reinforcement during second-order conditioning, in which a previously reinforced CS1 can act as US in subsequent conditioning of a second CS2. As Drosophila is capable of second-order learning, this theory can be tested in behavioral experiments: if associative strengthening of DAN responses to the CS underlies CS1-induced reinforcement in second-order conditioning, then preventing associative plasticity in DANs, or blocking their output during CS2-CS1 pairing should abolish second-order conditioning (Dylla, 2017).

    The occurrence of CS-induced responses and associative plasticity in most of the MB-innervating DANs suggests that the separation between the CS- and US-pathway and between different US-pathways is less strict than suggested in current models of associative learning in the MB. Associative plasticity in the spatial pattern of CS-induced DAN responses makes them a potential neuronal substrate for encoding the US identity in CS-US memories and the predictive power of a CS (Dylla, 2017).

    These data revealed similar response properties and plasticity rules across Drosophila DANs in the γ- and β'-lobe. This contrasts with their anatomical and functional heterogeneity, which indicates yet undiscovered mechanisms and functions of DAN plasticity. Note, that this study could not test whether the flies learned in the imaging setup, as currently no behavioral readout exists for odor-shock conditioning during physiological experiments. Nevertheless, since a conditioning protocol and stimulus application comparable to an established behavioral paradigm was used, it is believed that the associative plasticity in neuronal responses that was found underlies behavioral associative plasticity. Therewith the data lay the foundations for causal studies on the function of associative plasticity in DANs (Dylla, 2017).

    Neuroarchitecture of aminergic systems in the larval ventral ganglion of Drosophila melanogaster

    Biogenic amines are important signaling molecules in the central nervous system of both vertebrates and invertebrates. In the fruit fly Drosophila melanogaster, biogenic amines take part in the regulation of various vital physiological processes such as feeding, learning/memory, locomotion, sexual behavior, and sleep/arousal. Consequently, several morphological studies have analyzed the distribution of aminergic neurons in the CNS. Previous descriptions, however, did not determine the exact spatial location of aminergic neurite arborizations within the neuropil. The release sites and pre-/postsynaptic compartments of aminergic neurons also remained largely unidentified. This study used gal4-driven marker gene expression and immunocytochemistry to map presumed serotonergic (5-HT), dopaminergic, and tyraminergic/octopaminergic neurons in the thoracic and abdominal neuromeres of the Drosophila larval ventral ganglion relying on Fasciclin2-immunoreactive tracts as three-dimensional landmarks. With tyrosine hydroxylase- (TH) or tyrosine decarboxylase 2 (TDC2)-specific gal4-drivers, the distribution of ectopically expressed neuronal compartment markers was examined in presumptive dopaminergic TH and tyraminergic/octopaminergic TDC2 neurons, respectively. The results suggest that thoracic and abdominal 5-HT and TH neurons are exclusively interneurons whereas most TDC2 neurons are efferent. 5-HT and TH neurons are ideally positioned to integrate sensory information and to modulate neuronal transmission within the ventral ganglion, while most TDC2 neurons appear to act peripherally. In contrast to 5-HT neurons, TH and TDC2 neurons each comprise morphologically different neuron subsets with separated in- and output compartments in specific neuropil regions. The three-dimensional mapping of aminergic neurons now facilitates the identification of neuronal network contacts and co-localized signaling molecules, as exemplified for DOPA decarboxylase-synthesizing neurons that co-express crustacean cardioactive peptide and myoinhibiting peptides (Vömel, 2008).

    This study used gal4-driven marker gene expression and immunocytochemistry to three-dimensionally map presumed serotonergic, dopaminergic and tyraminergic/octopaminergic neurons within the Fas2 landmark system of the larval VG. Furthermore, several ectopically expressed pre- and postsynaptic markers were employed to reveal the in- and output compartments of presumptive dopaminergic TH and tyraminergic/octopaminergic TDC2 neurons. The results allow comparison of the segmental distribution patterns of aminergic neurons and to trace aminergic projections to defined neuropil areas within the VG. In the following, the morphology of aminergic neurons are related to known biogenic amine (BA) functions and describes putative neuronal network interactions with other VG neurons. This work also exemplifies how Fas2-based mapping can simplify the identification of co-localized signaling molecules, and allocate all neurons within the complex Ddc-gal4 expression pattern to distinct neuron subsets (Vömel, 2008).

    Throughout the insects, similar neuron groups synthesize BAs. These groups typically comprise only few neurons with large branching patterns. In agreement with previous studies, 5-HT neurons in t1-a8 of the Drosophila larval VG represent interneurons with intrasegmental neurites. The 5-HT neurons of a8, however, appear to supply only the neuropil of a7, but not that of a8 and the adjacent 'terminal plexus'. Like 5-HT neurons, the presumptive dopaminergic TH neurons lack peripheral projections and appear to exclusively represent interneurons. In contrast, presumptive tyraminergic/octopaminergic TDC2 neurons mostly represent efferent vumTDC2 neurons. The vumTDC2 neurons obviously project to larval body wall muscles including M1 and M2 since these muscles showed TA- and OA-immunoreactive type II boutons. In a8, dorsally located dmTDC2 neurons send axons through the associated segmental nerves, and hence are efferent neurons as well. These dmTDC2 neurons probably innervate the reproductive tract in the adult female fly. Besides the dmTDC2 neurons of a8, typically two additional dmTDC2 neurons reside in the dorsal cortex between the last subesophageal neuromere and t1. These dmTDC2 neurons were not described in previous morphological studies on TA- and OA-/TβH-immunoreactive neurons. Nevertheless, all dmTDC2 neurons in the VG consistently showed strong Tdc2-gal4-driven mCD8GFP expression as well as TßH immunoreactivity. Thus, they likely synthesize both TA and OA. Although their neurites could not be traced, the dmTDC2 neurons resemble a pair of anterior medial neurons in locusts and crickets that localize to t1 and innervate the anterior connectives. Alternatively, dmTDC2 neurons may correspond to a single dorsal unpaired median neuron which resides in t1 of the locust and supplies the subesophageal nerves. Like dmTDC2 neurons, pmTDC2 neurons are probably interneurons as well. The soma position of pmTDC2 neurons highly resembles that of descending OA-immunoreactive interneurons detected in the subesophageal and thoracic neuromeres of bees, crickets, cockroaches, locusts, and moths (Vömel, 2008).

    Within the larval VG of Drosophila, aminergic neurons typically show a segmentally reiterated distribution. The number of aminergic modules, however, often varies between different neuromeres. 5-HT neurons, for instance, typically occur as two bilateral pairs per neuromere. Yet, t1 comprises three 5-HT neuron pairs and a8 only one pair. The presumptive dopaminergic TH neurons also lack a strict serial homology since three ventral median TH (vmTH) neurons are present in t1, but only one in t2-a7. Furthermore, dlTH neurons locate to a1-7, but appear to be missing in t1-3. The neuromere a8 lacks TH neurons. The number of presumptive tyraminergic/octopaminergic TDC2 neurons differs between various neuromeres as well. Whereas t1 comprises one or two dmTDC2 neurons, comparable neurons are absent in t2-a7. Putative descending pmTDC2 interneurons localize to t1-a1, but appear to be missing in the remaining abdominal neuromeres. Taken together, the number of aminergic modules in t1 and a8 often deviated from that of t2-a7. This difference may-at least partially-reflect unique neuronal circuits in t1 and a8. While t1 specific physiological functions in larvae are unknown, a8 and the adjacent 'terminal plexus' are associated with the tail region, and hence contain a specific set of sensory neurons and motoneurons. The terminal neuromeres also supply several unique structures such as the spiracles or the anal pads (Vömel, 2008).

    Besides the segmental differences in neuron number, the density of aminergic innervation and the amount of immunolabeling/marker gene expression varies between neuromeres as well. In particular, presumptive dopaminergic TH neurons show a striking neuromere-specific labeling pattern. Whereas a1-5 contain only few labeled TH projections, t1-3 and a6-7 comprise a comparably dense network of TH neurites. Similar to TH neurons, 5-HT neurons most densely innervate the neuropil of a7. Since a high extracellular concentration of 5-HT decreases the density of 5-HT-immunoreactive arborizations within the neuropil, a7 may represent a minor 5-HT release site. In contrast to a7, the neuropil of a8 and the adjacent 'terminal plexus' (which receive prominent peptidergic innervation) typically lack aminergic neurite arborizations. Consequently, larval aminergic neurons may play a subordinate role in tail-related physiological processes (Vömel, 2008).

    To reveal putative synaptic in- and output zones of aminergic neurons, the neuronal compartment markers neuronal synaptobrevin-GFP, synaptotagmin 1-GFP, and Drosophila Down syndrome adhesion molecule [17.1]-GFP were employed. Neuronal synaptobrevin is a vesicle associated membrane protein that plays a role in the SNARE complex during vesicle transport and fusion with the plasma membrane. In accordance with this function, ectopically expressed neuronal synaptobrevin-GFP (SybGFP) accumulates at nerve terminals. SybGFP therefore served to define the presynaptic compartments of several Drosophila neurons, e.g. in the visual system. However, neuronal synaptobrevin is not restricted to small synaptic vesicles, but also locates to the membrane of large dense core vesicles, which contain BAs or neuropeptides. Consequently, in a7, SybGFP localized to putative release sites of presumptive serotonergic DDC neurons. SybGFP was also used to identify non-synaptic release sites in several peptidergic neurons. In aminergic neurons, the distribution of gal4-driven SybGFP highly resembled the corresponding mCD8GFP expression pattern. SybGFP localized in dotted patterns to aminergic neuron somata and associated neurites. It is therefore suggested that SybGFP does not exclusively label the presynaptic compartments of aminergic neurons. This fits to the assumption that ectopically expressed synaptic proteins can either localize to transport vesicles or non-synaptic compartments in peptidergic neurons. On the other hand, the ubiquitous distribution of SybGFP in aminergic neurites may suggest a widespread BA release/recycling from non-synaptic active sites. In mammals, BA release/recycling is not restricted to synapses. Vesicular monoamine transporters, which transport BAs into secretory vesicles, reside within neuron somata, axons, and dendrites. In Drosophila, the vesicular monoamine transporter DVMAT-A localizes to somata as well as neurites of several aminergic neurons both in the larval. Thus, the widespread distribution of SybGFP and DVMAT-A in aminergic neurons suggests that a considerable amount of aminergic vesicles resides at non-synaptic sites. Non-synaptic BA release/recycling might therefore play a major role for aminergic neuronal network signaling (Vömel, 2008).

    Like neuronal synaptobrevin, synaptotagmins also represent integral membrane proteins of both small synaptic and large dense core vesicles. In Drosophila, the products of seven synaptotagmin genes localize to distinct neuronal compartments including the postsynaptic site. At the presynaptic site, synaptotagmin 1 does not participate in the SNARE complex, but acts as a Ca2+-sensor for synaptic vesicle fusion. Furthermore, synaptotagmin 1 appears to be the only crucial isoform for synaptic vesicle release. Consequently, a synaptotagmin 1-GFP fusion construct (SytGFP) was developed as a synaptic vesicle marker that specifically labels presynaptic sites. In aminergic neurons, the distribution pattern of SytGFP strikingly differed from the observed mCD8GFP and SybGFP labeling. Primary neurites of aminergic neurons always completely lacked SytGFP. Varicose neurite structures which were less evident in the mCD8GFP and SybGFP expression patterns showed strong SytGFP labeling. In agreement with the SytGFP distribution in other Drosophila neuron types, SytGFP hence appears to exclusively accumulate at the presynaptic sites of aminergic neurons. Thus, SytGFP represents a valuable marker to separate synapses from other neuronal compartments in aminergic neurons. However, since BA release is not restricted to synapses, SytGFP may not label all BA release sites of aminergic neurons. The sparse co-localization of SytGFP and SybGFP in aminergic neurites in fact suggests that aminergic vesicles-which are located distal to presynaptic sites-generally lack SytGFP. Consequently, non-synaptic BA release appears to be independent of synaptotagmin 1, but may depend on other synaptotagmin isoforms such as synaptotagmin α or β. The differing distribution of SytGFP and SybGFP also suggests that aminergic neurons contain several types of aminergic vesicles which are either associated with presynaptic or non-synaptic BA release. Alternatively, aminergic neurons may synthesize additional non-aminergic neurotransmitters like acetylcholine, GABA, or glutamate. Presumed octopaminergic efferent neurons, for instance, appear to release glutamate from type II terminals at the neuromuscular junction. In such neurons, SytGFP likely labels presynaptically located transmitter vesicles and may not reveal BA release sites (Vömel, 2008).

    In contrast to SybGFP and SytGFP, ectopically expressed Drosophila Down syndrome adhesion molecule [17.1]-GFP (DscamGFP) localized to postsynaptic compartments and not to axons or presynaptic sites. Consequently, DscamGFP has served as dendrite marker in mushroom body lobe neurons. Aminergic neurons showed only weak DscamGFP labeling. DscamGFP primarily localized to neurites that lacked SytGFP labeling. Since SytGFP accumulates at presynaptic sites, DscamGFP appears to represent a valuable marker to define dendritic compartments in aminergic neurons (Vömel, 2008).

    In 5-HT neurons, the distribution of ectopically expressed neuronal compartment markers was not examined since specific gal4 drivers are not available. The Ddc-gal4 driver induces marker gene expression not only in presumed serotonergic, but also in dopaminergic and additional peptidergic neurons. Consequently, neurites of different DDC neuron subsets overlap in specific neuropil areas. Presumptive serotonergic as well as dopaminergic DDC neurites, for instance, localize to the VG neuropil above the CI tracts. These conditions prevent an accurate description and interpretation of the compartment marker distribution in presumptive serotonergic DDC neurons. Thus, appropriate gal4 drivers (e.g. Dtph-gal4) are needed to further analyze 5-HT neuron morphology (Vömel, 2008).

    5-HT neurons bifurcate strongly in the whole neuropil of t1-a7, and hence may influence various VG neurons including sensory, inter- as well as motoneurons. However, putative neuronal network contacts of 5-HT neurons were not examined since previous morphological studies on Drosophila 5-HT receptors did not describe the exact spatial location of the respective receptors in the larval VG (Vömel, 2008).

    In TH neurons, the distribution of ectopically expressed mCD8GFP, SybGFP, SytGFP and DscamGFP differed only slightly. This might relate to the fact that the VG contains two different TH neuron groups, the vmTH and dlTH neurons, whose neurites contact each other at longitudinal projections. Consequently, pre- and postsynaptic compartments of both TH neuron groups appeared to overlap, e.g. at longitudinal projections next to the VL tracts. Since additional TH neurons located in the brain or subesophageal ganglia also innervate the VG, it was not possible to clarify which TH neuron group attributes to a particular neuronal projection. Several morphological findings, however, suggest that TH neurons possess distinct in- and output sites: Most strikingly, a1-5 contained less TH neurites labeled with mCD8GFP, SybGFP and DscamGFP, as compared to t1-3 and a6-7. In t1-a7, high amounts of SybGFP and SytGFP located to lateral longitudinal projections next to the VL tracts. These longitudinal TH neurites also contained a comparably high amount of DscamGFP, and hence likely represent synaptic in- as well as output compartments of different TH neuron groups. Besides lateral longitudinal TH projections, SybGFP and SytGFP also co-localized to the median neuropil between the DM/VM tracts. At least in a1-5, this neuropil area lacked DscamGFP, and hence probably represents a presynaptic output site of TH neurons. In a6-7, a comparably strong SybGFP and SytGFP labeling was observed in arborizations around transversal TH neurites. Whereas SybGFP mainly located to the dorsal branches of the transversal TH neurite loops, SytGFP and DscamGFP primarily labeled the ventral branches. Thus, the dorsal branches of the transversal TH neurite loops may represent non-synaptic DA release sites, while the ventral branches seem to comprise overlapping synaptic in- and output compartments of different TH neuron groups (Vömel, 2008).

    Both vmTH and dorso-lateral TH (dlTH) neurons innervate distinct neuropil areas within the VG. The vmTH neurons send their primary neurites dorsally and then project through the dorsal part of the neuropil above Transversal projection (TP) 3. Since the dorsal neuropil comprises the dendritic compartments of most motoneurons, vmTH neurites are ideally located to modulate locomotor activity. This fits to the finding that DA application onto intact larval CNS-segmental preparations rapidly decreased the rhythmicity of CNS motor activity and synaptic vesicle release at the neuromuscular junction. Unlike vmTH neurons, dlTH neurons exclusively innervate the ventral part of the VG neuropil beneath TP 3. There, putative dendritic compartments of TH neurons mainly localize to lateral longitudinal and to transversal projections adjacent to the main output site of several afferent sensory neurons, e.g. tactile and proprioreceptive neurons. Thus, some TH neurons may receive synaptic input from specific sensory neurons. In contrast, TH neurons also seem to have output sites in the ventral part of the neuropil, and hence may influence the signal transmission between sensory neurons and interneurons. This fits to the finding that peptidergic apterous neurons, which appear to transmit sensory input from the VG to the brain, express DA receptors. Concomitantly, dendritic compartments of apterous neurons seem to reside adjacent to the putative DA release sites of TH neurons at the CI tracts. Besides the overlap between transversal TH neurites and sensory/interneuron projections in the ventral neuropil, TH neurons may influence several neuron groups at other locations within the VG. For instance, the putative synaptic output sites of TH neurons in the median neuropil between the DM/VM tracts overlap with presumptive input compartments of both interneurons and efferent neurons expressing peptides such as CCAP, corazonin, FMRFa, or MIP. Furthermore, the putative output sites at longitudinal TH projections next to the VL tracts lay adjacent to presumptive input compartments of e.g. efferent leucokininergic neurons (Vömel, 2008).

    In the VG, most TDC2 neurons are efferent vumTDC2 neurons and showed a differential distribution of ectopically expressed SybGFP, SytGFP, and DscamGFP. The primary neurites and transversal projections of vumTDC2 neurons were labeled with DscamGFP, but lacked SytGFP. Therefore, these neurites likely represent dendritic input sites. This fits to the finding that vumTDC2 neurons possess output sites at larval body wall muscles. However, vumTDC2 neurites within the VG also contained high amounts of SybGFP, and hence may release TA/OA from non-synaptic sites. Besides vumTDC2 neurites, SybGFP strongly labeled longitudinal TDC2 neurites and associated arborizations in the dorso-lateral neuropil between TP 1 and 3. These TDC2 projections showed prominent SytGFP labeling and TßH immunoreactivity, but largely lacked DscamGFP. Thus, the dorsal part of the VG neuropil likely contains output compartments of TDC2 neurons. Since the larval brain seems to contain only tyramine- and no octopamine-immunoreactive neurons, these output sites likely derive from descending interneurons located in the subesophageal ganglia, dmTDC2 or pmTDC2 neurons. Noteworthy, the strong SybGFP and SytGFP labeling in TDC2 neurites projecting through the dorso-lateral neuropil of the VG overlapped with DscamGFP in transverse vumTDC2 neurites. Thus, descending TDC2 neurons may interact with vumTDC2 neurons (Vömel, 2008).

    The VG comprises efferent vumTDC2 neurons as well as several putative TDC2 interneuron groups. Since all vumTDC2 neurons appear to have synapses at peripheral targets and dendrites in the dorsal neuropil, they show the typical motoneuron morphology. This corresponds to the finding that OA inhibited synaptic transmission at the neuromuscular junction by affecting both pre- and postsynaptic mechanisms. In addition, T?H mutant larvae, with altered levels of TA and OA, showed severe locomotion defects, which seemed to be linked to an imbalance between TA and OA signaling. Hence, vumTDC2 neurons likely regulate peripheral processes such as body wall muscle activity, whereas TDC2 interneurons centrally modulate the neuronal activity of motoneurons and interneurons involved in locomotor control. Interestingly, presumptive presynaptic compartments of descending TDC2 interneurons reside adjacent to transversal vumTDC2 dendrites. Thus, both TDC2 neuron groups may interact to modulate larval locomotor activity. Besides their function for locomotion, descending TDC2 neurons may also influence other neurons which project into the dorsal neuropil between TP 1 and 3. The putative output sites of TDC2 interneurons, for instance, lay adjacent to several peptidergic projections showing allatostatin-A, FMRFa, MIP or tachykinin immunoreactivity. However, nothing is known about TA/OA receptor distribution in the larval VG (Vömel, 2008).

    During the morphological analysis of DDC neurons in the L3 larval VG, two DDC neuron groups were identified that obviously synthesize neither 5-HT nor DA. This corresponds to the previous finding that Ddc-gal4-driven marker gene expression is not restricted to presumptive serotonergic 5-HT and dopaminergic TH neurons. However, it cannot be excluded that the putative non-aminergic DDC neurons transiently synthesize BAs during other developmental stages. Ddc-gal4-driven mCD8GFP expression never revealed the dlTH neurons. This may relate to the fact that the onset of Ddc expression varies between different DDC neuron groups, and high DDC and TH levels do not temporally coincide. Taken together, these results suggest that-at least in the L3 larval VG-the Ddc-gal4 expression pattern 1) contains additional non-aminergic neurons, and 2) typically comprises most, but not all 5-HT and TH neurons. These particular characteristics of the Ddc-gal4 driver line should be carefully considered for the interpretation of studies that employed Ddc-gal4-driven expression to genetically manipulate serotonergic or dopaminergic neurons. Nevertheless, since all Ddc-gal4 expressing neurons within the VG showed at least faint DDC immunoreactivity, the Ddc-gal4 driver appears to restrict ectopical gene expression to DDC neurons. Noteworthy, the DDC neurons which lacked 5-HT and TH immunoreactivity showed corazonin and CCAP/MIP immunoreactivity respectively. In the moth Manduca sexta, these peptides play vital roles during ecdysis. At least the CCAP/MIP neurons are also necessary for the proper timing and execution of ecdysis behavior in Drosophila. Since dopaminergic DDC neurons regulate the titers of the molting hormones 20-hydroxyecdyson and juvenile hormone, both aminergic and peptidergic DDC neurons may interact to control ecdysis-related events. Recent findings indeed suggest that CCAP/MIP neurons modulate TH activity after eclosion to control the precise onset of tanning (Vömel, 2008).

    Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis

    In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). This study shows that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, β3-octopamine receptor (Octβ3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octβ3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulated ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. The study proposes that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant (Ohhara, 2015).

    In many animal species, the developmental transition is a well-known biological process in which the organism alters its body morphology and physiology to proceed from the juvenile growth stage to the adult reproductive stage. For example, in mammals, puberty causes a drastic change from adolescent to adulthood, whereas in insects, metamorphosis initiates alteration of body structures to produce sexually mature adults, a process accompanied by changes in habitat and behavior. These developmental transitions are primarily regulated by steroid hormones, production of which is regulated coordinately by developmental timing and nutritional conditions. How these processes are precisely regulated in response to developmental and environ mental cues is a longstanding question in biology (Ohhara, 2015).

    In holometabolous insects, the steroid hormone ecdysone plays a pivotal role in metamorphosis. In Drosophila, metamorphic development from the third-instar larva into the adult, through the prepupa and pupa, initiates 90-96 h after hatching (hAH) at 25°C under a standard culture condition. At the onset of the larval-prepupal transition, ecdysone is produced in the prothoracic gland (PG) and then converted into its active form, 20-hydroxyecdysone (20E), in the peripheral organs. The activities of 20E terminate larval development and growth and initiates metamorphosis. Ecdysone biosynthesis is regulated in the PG by neuropeptides, enabling modulation of the timing of 20E pulses during development. The best-known stimulator of ecdysone biosynthesis is prothoracico-tropic hormone (PTTH), which is produced by neurons in the CNS. PTTH activates the receptor tyrosine kinase Torso in the PG to stimulate expression of ecdysone biosynthetic genes through the Ras85D/Raf/MAPK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Insulin-like peptides (Ilps), members of another class of neuron-derived factors, activate PI3K in the PG, resulting in production of ecdysone biosynthetic proteins. The Activin/transforming growth factor-β (TGF-β) signaling pathway is also required in the PG for the expression of PTTH and Ilps receptors, although to date it remains unclear which organ produces the ligand that acts on the PG (Ohhara, 2015).

    In addition to these neuropeptides, the larval-prepupal transition is modulated by environmental cues such as nutritional conditions that influence larval body size. For example, at 56 hAH, early third-instar larvae attain the minimal viable weight (MVW), at which sufficient nutrition is stored in larvae to ensure their survival through metamorphosis. After attaining MVW, larvae pass another checkpoint, critical weight (CW), defined as the minimum larval size at which starvation no longer delays the larval-prepupal transition. In Drosophila, both checkpoints occur almost simultaneously, making it difficult to distinguish them. However, CW is regarded as a body size checkpoint that initiates metamorphosis and is therefore believed to ultimately modulate ecdysone production in the PG. However, its downstream effectors and signaling pathway remain elusive (Ohhara, 2015).

    Based on data obtained in Manduca and Bombyx, a G protein-coupled receptor (GPCR) has long been postulated to be essential for ecdysone biosynthesis in the PG. However, this GPCR and its ligand have not yet been identified. This study shows that monoaminergic autocrine signaling through a GPCR, β3-octopamine receptor (Octβ3R), plays an essential role in ecdysone biosynthesis to execute the larval-prepupal transition. Octβ3R is also required for activation of PTTH and Ilps signaling. It is proposed that this autocrine system acts downstream of the CW checkpoint to allow the larval-prepupal transition. It is speculated that monoamines play an evolutionarily conserved role in the regulation of steroid hormone production during developmental transitions (Ohhara, 2015).

    Previously studies have shown that the GPCR Octβ3R is expressed in multiple larval tissues, including the PG. To determine whether Octβ3R is involved in ecdysone biosynthesis and metamorphosis, RNAi was used to knock down Octβ3R function specifically in the PG, using the Gal4-upstream activation sequence (UAS) system. Two different UAS-Octβ3RRNAi constructs targeting distinct regions of the Octβ3R mRNA (Octβ3RRNAi-1 and Octβ3RRNAi-2) were used to knock down Octβ3R in the PG with the help of a phantom (phm)-22-Gal4 driver. Strikingly, larvae expressing Octβ3RRNAi in the PG never developed into adult flies, and 96% of phm>Octβ3RRNAi-1 animals and 34% of phm>Octβ3RRNAi-2 animals arrested at the larval stage. When UAS-dicer2 was introduced into phm>Octβ3RRNAi-2 larvae (phm>Octβ3RRNAi-2+dicer2) to increase RNAi activity, all of these animals arrested at the larval stage. Using in situ hybridization, a significant reduction was confirmed in the Octβ3R mRNA levels in the PG of knockdown animals relative to control larvae. These data suggest that Octβ3R expression in the PG is essential for executing the larval-prepupal transition in metamorphosis (Ohhara, 2015).

    Because a similar defect in the larval-prepupal transition occurs in ecdysone-deficient larvae, it was hypothesized that the Octβ3R knockdown phenotype was due to lack of ecdysone production. Consistent with this idea, the 20E titer was much lower in phm>Octβ3RRNAi-1 larvae than in control larvae just before the larval-prepupal transition (90 hAH). Moreover, administration of 20E by feeding rescued the defect in the larval- prepupal transition caused by Octβ3R knockdown. When phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae were cultured on media containing 20E (1 mg/mL) from 48 hAH onward, approximately half of them developed to the prepupal stage, compared with only 2-3% of larvae not fed 20E. Thus, PG-specific loss of Octβ3R activity causes an arrest in the larval-prepupal transition due to lack of ecdysone (Ohhara, 2015).

    Ecdysone is synthesized in the PG from dietary cholesterol through the action of seven ecdysone biosynthetic genes (neverland, spookier, shroud, Cyp6t3, phantom, disembodied, and shadow). Quantitative RT- PCR (qPCR) was performed to investigate whether loss of Octβ3R function affects expression of these genes in the PG. In control larvae, expression of these genes increased dramatically between 72 and 96 hAH, when the larval-prepupal transition occurs. By contrast, in phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae, the expression of all of these genes was significantly reduced relative to control larvae at 96 hAH. The reduced expression of ecdysone biosynthetic genes in the PG was confirmed by in situ hybridization. Furthermore, immunostaining revealed that Neverland, Shroud, Phantom, Disembodied, and Shadow protein levels were reduced in the PG of phm>Octβ3RRNAi-1 and phm>Octβ3RRNAi-2+dicer2 larvae. Taken together, these data show that Octβ3R function is required in the PG for proper expression of ecdysone biosynthetic genes (Ohhara, 2015).

    Octβ3R is thought to be activated by octopamine and tyramine binding. Octopamine is synthesized from tyramine by tyramine β-hydroxylase (Tbh), and tyramine is synthesized from tyrosine by tyrosine decarboxylase (Tdc). In Drosophila, two Tdc genes (Tdc1 and Tdc2) and one Tbh gene have been identified, and all of them are expressed in the larval CNS. Tdc1, Tdc2, and Tbh are also expressed in the PG. Furthermore, octopamine and tyramine were detected in the PG by immunostaining. Thus, octopamine and/or tyramine synthesized in the PG may activate Octβ3R in an autocrine manner to induce ecdysone production (Ohhara, 2015).

    To test this, PG-specific knockdowns of Tdc1, Tdc2, and Tbh were generated. To knock down Tdc2, two constructs targeting distinct regions of the Tdc2 transcript (Tdc2RNAi-1 and Tdc2RNAi-2) were expressed along with dicer2 in the PG under the control of the phm-22-Gal4 driver (phm > Tdc2RNAi-1+dicer2 and phm > Tdc2RNAi-2+dicer2). All phm > Tdc2RNAi-1+dicer2 larvae arrested at the larval stage, and phm > Tdc2RNAi-2+dicer2 larvae were significantly delayed at the larval-prepupal transition, relative to control animals. Tdc2 mRNA level was reduced in the ring gland (RG) containing the PG in both sets of knockdown animals, as demonstrated by qPCR. Moreover, octopamine and tyramine production in the PG was impaired by Tdc2 knockdown. By contrast, Tdc1 knockdown (phm > Tdc1RNAi+dicer2) caused only a subtle delay in the larval-prepupal transition and had no detectable effect on octopamine or tyramine production. These results suggest that Tdc2 is the predominant Tdc regulating octopamine and tyramine biosynthesis in the PG and the larval-prepupal transition. Contrary to these findings, a null mutation in Tdc2 does not affect metamorphosis, and these mutant flies are viable. Thus, PG-specific knockdown causes a stronger phenotype than complete loss of Tdc2 activity in whole animals. A similar situation has been reported in regulation of metamorphosis by Activin signaling. These phenomena can be explained by a model in which some compensatory changes in other mutant tissues rescue the PG-specific knockdown phenotype in null-mutant animals (Ohhara, 2015).

    PG-specific Tdc2 knockdown caused a reduction in larval 20E concentration. Therefore, whether feeding 20E to Tdc2 knockdown larvae would rescue the larval- prepupal transition defect was examined. To this end, phm > Tdc2RNAi-1+ dicer2 and phm > Tdc2RNAi-2+dicer2 larvae were cultured in media with or without 20E (1 mg/mL) from 48 hAH onward. Approximately 40% of the 20E-fed phm > Tdc2RNAi-1+dicer2 larvae developed to the prepupal stage, whereas none of those larvae grown on control media progressed beyond the larval stage. Furthermore, the delay in the larval-prepupal transition in phm > Tdc2RNAi-2+dicer2 larvae was rescued by 20E feeding. These results indicate that the defect in the larval-prepupal transition in Tdc2 knockdown animals results from a lack of 20E production. Thus, octopamine/ tyramine synthesized in the PG appears to activate Octβ3R in an autocrine manner to execute the larval-prepupal transition by regulating ecdysone production (Ohhara, 2015).

    To determine which Octβ3R ligand is responsible for this autocrine signaling, Tbh was knocked down in the PG to prevent conversion of tyramine into octopamine. To knock down Tbh, two constructs targeting distinct regions of the Tbh transcript (TbhRNAi-1 and TbhRNAi-2) were expressed along with dicer2 under the control of phm-22-Gal4 (phm > TbhRNAi-1+ dicer2 and phm > TbhRNAi-2+dicer2). Although the Tbh knockdown caused a reduction in octopamine production in the PG, these larvae did not exhibit any obvious defects in the larval-prepupal transition or subsequent metamorphosi. These data suggest that tyramine, rather than octopamine, is the Octβ3R ligand that activates ecdysone production in the PG (Ohhara, 2015).

    Because ecdysone biosynthesis in the PG is under the control of Ilps and PTTH signaling, it was next examined whether Octβ3R function is required to activate these signaling pathways. To detect Ilps signaling activity, a pleckstrin-homology domain fused to GFP (PH-GFP), which is recruited to the plasma membrane when insulin signaling is activated, was used. In the PG cells of control larvae, PH-GFP was only weakly localized to the plasma membrane at 48 hAH, whereas its membrane localization became increasingly evident at 60, 84, and 90 hAH. By contrast, in PG cells of phm>Octβ3RRNAi-1 larvae, the tight localization of PH-GFP to the plasma membrane was no longer detectable, indicating that activation of Ilps signaling had been disrupted. Moreover, overexpression of a constitutively active form of the Ilps receptor InR (InRCA) was able to rescue the larval arrest in phm>Octβ3RRNAi-1 animals. Next, immunostaining was performed of the diphosphorylated form of ERK (dpERK), a downstream signaling component of the PTTH pathway. dpERK expression was found to be very weak at 48 hAH, but was activated in the PG of control larvae at 60, 84, and 90 hAH; by contrast, this activation was reduced in the PG of phm>Octβ3RRNAi-1 larvae. Expression of a constitutively active form of another downstream PTTH signaling component, Ras (RasV12), rescued the larval-prepupal transition defect in phm>Octβ3RRNAi-1 animals. These results show that Octβ3R function is required to activate Ilps and PTTH signaling in the PG and that these signaling pathways execute the larval-prepupal transition. Although activation of both the Ilps and PTTH signaling pathways requires Activin/TGFβ signaling in the PG, expression of a constitutively active form of the Activin/ TGFβ receptor Baboon (BaboCA) failed to rescue the larval-prepupal transition defect in phm>Octβ3RRNAi-1 animals. This observation suggests that Octβ3R acts downstream or independent of Activin/TGFβ signaling to regulate Ilps and PTTH signaling in the PG (Ohhara, 2015).

    The observations described above demonstrate that phm>Octβ3RRNAi affects Ilps and PTTH signaling in the PG as early as 60 hAH, raising the question of when Octβ3R function is required in the PG for execution of the larval-prepupal transition. To address this issue, the Gal80ts and Gal4/UAS system, which restricts expression of Octβ3R dsRNA in the PG at 18oC, but allows its expression at 28oC, was used. The results of temperature upshift and downshift experiments revealed that the larval-prepupal transition was impaired only when Octβ3R dsRNA was expressed in the PG at around 60 hAH. Notably, 60 hAH is the critical period during which larvae attain CW under nutrient-rich conditions. As noted above, when larvae are starved before attainment of CW, they are unable to transit into the prepupal stage. By contrast, starved larvae can successfully transit to prepupal/pupal stage without developmental delay once they have attained CW by growing beyond the critical period (~56 hAH) under nutrient-rich conditions in standard Drosophila medium. Thus, it is hypothesized that Octβ3R signaling acts downstream of the body-size checkpoint, or attainment of CW, to allow the larval-prepupal transition (Ohhara, 2015).

    Several lines of evidence support this hypothesis. First, Octβ3R function is required for activation of Ilps and PTTH signaling detected in the PG at 60 hAH. By contrast, at 48 hAH, before the attainment of CW, neither signaling pathway is active in the PG. Second, Ilps and PTTH signaling was not activated in the PG when the larvae were starved from 48 hAH onward (early starvation), whereas these signaling pathways were active when the larvae were starved after 60 hAH (late starvation). Finally, a ligand for Octβ3R, tyramine, was detectable in the PG at 60 hAH, but decreases after this stage under a nutrient-rich condition. This decrease in tyramine was abrogated by early starvation but not by late starvation. Assuming that this decrease in tyramine in the PG is due to its secretion from PG cells, it is reasonable to propose that attainment of CW causes tyramine secretion from the PG at around 60 hAH, which in turn activates Octβ3R to regulate the Ilps and PTTH pathways, leading to the larval-prepupal transition (Ohhara, 2015).

    This study demonstrates that monoaminergic regulation plays a pivotal role in ecdysone biosynthesis to induce metamorphosis and that Octβ3R acts as an upstream regulator essential for the Ilps and PTTH signaling. In addition, the data indicate that Octβ3R ligands are produced in the PG to stimulate ecdysone biosynthesis in an autocrine manner. Autocrine signaling has been proposed to mediate the community effect, in which identical neighboring cells are coordinated in their stimulation and maintenance of cell type-specific gene expression and their differentiation, as observed in muscle development of amphibian embryos. Thus, it is proposed that monoaminergic autocrine signaling among PG cells acts to increase their responsiveness to Ilps and PTTH, thereby allowing coordinated ex- pression of ecdysone biosynthetic genes within a time window following exposure to neuropeptides (Ohhara, 2015).

    These findings raise the larger question of whether monoamine acts as part of an evolutionarily conserved mechanism of steroid hormone production. In vertebrates, there is limited evidence of monoaminergic regulation of steroid hormone biosynthesis. For example, in cultured adrenal glands, catecholamine stimulates the biosynthesis of the steroid hormone cortisol in a paracrine manner to elicit a stress reaction. Another example is the Leydig cells of the mammalian testes, in which the steroid hormone testosterone is produced mainly in response to pituitary gonadotropin. However, catecholamine signaling through β-adrenergic receptors, the orthologs of Octβ3R, also promotes the production of testosterone from cultured fetal Leydig cells, which may be the site of catecholamine synthesis in the fetal and mature human testes. Thus, monoamines may play a conserved role in modulating and/or stimulating steroid hormone production during physiological and developmental transitions (Ohhara, 2015).

    Limited distal organelles and synaptic function in extensive monoaminergic innervation

    Organelles such as neuropeptide-containing dense-core vesicles (DCVs) and mitochondria travel down axons to supply synaptic boutons. DCV distribution among en passant boutons in small axonal arbors is mediated by circulation with bidirectional capture. However, it is not known how organelles are distributed in extensive arbors associated with volume transmission and neuromodulation by monoamines and neuropeptides and mammalian dopamine neuron vulnerability. Therefore, this study examined presynaptic organelle distribution in Drosophila octopamine neurons that innervate approximately 20 muscles with approximately 1500 boutons. Unlike in smaller arbors, distal boutons in these arbors contain fewer DCVs and mitochondria, although active zones are present. Absence of vesicle circulation is evident by proximal nascent DCV delivery, limited impact of retrograde transport and older distal DCVs. Traffic studies show that DCV axonal transport and synaptic capture are not scaled for extensive innervation, thus limiting distal delivery. Activity-induced synaptic endocytosis and synaptic neuropeptide release are also reduced distally. It is proposed that limits in organelle transport and synaptic capture compromise distal synapse maintenance and function in extensive axonal arbors, thereby affecting development, plasticity and vulnerability to neurodegenerative disease (Tao, 2017).

    Octopamine mediates starvation-induced hyperactivity in adult Drosophila

    Starved animals often exhibit elevated locomotion, which has been speculated to partly resemble foraging behavior and facilitate food acquisition and energy intake. Despite its importance, the neural mechanism underlying this behavior remains unknown in any species. This study confirmed and extended previous findings that starvation induced locomotor activity in adult fruit flies Drosophila melanogaster. It was also shown that starvation-induced hyperactivity was directed toward the localization and acquisition of food sources, because it could be suppressed upon the detection of food cues via both central nutrient-sensing and peripheral sweet-sensing mechanisms, via induction of food ingestion. It was further found that octopamine, the insect counterpart of vertebrate norepinephrine, as well as the neurons expressing octopamine, were both necessary and sufficient for starvation-induced hyperactivity. Octopamine was not required for starvation-induced changes in feeding behaviors, suggesting independent regulations of energy intake behaviors upon starvation. Taken together, the current results establish a quantitative behavioral paradigm to investigate the regulation of energy homeostasis by the CNS and identify a conserved neural substrate that links organismal metabolic state to a specific behavioral output (Yang, 2015).

    Upon starvation, animals exhibit increased feeding and foraging behaviors, which in turn increases energy intake and restores energy homeostasis. The neural mechanism of feeding behavior, particularly how feeding is regulated by metabolic signals, has been extensively studied in both rodents and insect species. In contrast, how organismal metabolism influences foraging remains largely unclear (Yang, 2015).

    This study aimed to establish a behavioral paradigm for foraging behavior and to study how it is regulated by starvation. It has been reported that starvation induces hyperactivity in both rodents and fruit flies, but its relationship with foraging was unclear. It was first confirmed that starvation induced a robust and sustained increase in locomotion in adult fruit flies. Furthermore, it was found that starvation-induced hyperactivity could be suppressed upon the detection of nutritious substrate via an internal energy sensor SLC5A11, as well as the detection of food palatability via sweet-sensing gustatory neurons expressing Gr5a and Gr64a. It was also shown that although food intake per se was not sufficient to drive the suppressive effect on locomotion, nutrient/sweetness-induced fluid ingestion was indeed required. In addition, the data showed that mutant flies with a deficit in starvation-induced hyperactivity took longer time to locate and occupy desired food sources. Collectively, it is concluded that starvation-induced hyperactivity in adult flies resembles foraging behavior in laboratory conditions, because it is directed toward and facilitates the localization and acquisition of food. This set of behavioral assays therefore offers a platform and an entry point to further dissect the neural basis of this evolutionarily conserved and critical behavior (Yang, 2015).

    By using the foraging assay described above, octopamine, the insect counterpart of vertebrate norepinephrine, was found to be both necessary and sufficient for starvation-induced hyperactivity. In fruit flies, octopamine is only synthesized and released in a small number of CNS neurons (~100-150 neurons per fly). Therefore, the findings presented in this study offer a clear entry point to further dissect the neural circuitry that underlies foraging behavior in fruit flies. In addition, it is of obvious interest to examine whether norepinephrine is also involved in locomotor responses to starvation in rodents, such as FAA (Yang, 2015).

    Tyramine is the precursor of octopamine synthesis and itself can also function as a neurotransmitter in insects. In the present study, the possibility was exclued that tyramine alone was either necessary or sufficient for starvation-induced hyperactivity. However, the possibility was not excluded that octopamine and tyramine work in a synergistic way to regulate this behavior. Fruit flies express several receptors that are sensitive to both octopamine and tyramine in vitro, which serves as a potential 'hub' to integrate octopamine and tyramine signaling in vivo. Future research is needed to clarify the role of octopamine and tyramine in starvation-induced hyperactivity (Yang, 2015).

    Octopamine plays an important role in the regulation of a variety of fly behaviors, such as sleep, learning, and aggression. It is of interest to investigate whether and how different subsets of octopaminergic neurons modulate different behaviors in flies. Meanwhile, it is noteworthy that many of these behaviors regulated by octopamine signaling have a locomotor component. It is therefore plausible that the octopamine system may function as a general 'arousal' center, modulating physical activity of flies in response to external/internal cues such as circadian rhythm, conspecific chemosensory stimuli, and metabolic signals (Yang, 2015).

    Despite its importance in starvation-induced hyperactivity, this study found that octopamine was not required for starvation-induced changes in feeding behavior. These findings argue for independent regulations of multiple starvation-induced behavioral responses. Consistent with this idea, rodent studies have shown that several aspects of foraging behavior (e.g., FAA and food hoarding) do not require the genes and brain regions that are important for feeding control. In contrast, activating hypothalamic neurons expressing neuropeptide Y and agouti-related peptide promotes both feeding and locomotion, suggesting that feeding and foraging pathways converge at some point. It is therefore important to study whether there exists a common 'hunger' center in the CNS that coordinates various starvation-induced behaviors or whether different neural mechanisms independently sense changes in the metabolic state and modulate energy homeostasis (Yang, 2015).

    Octopamine controls starvation resistance, life span and metabolic traits in Drosophila

    The monoamines octopamine (OA) and tyramine (TA) modulate numerous behaviours and physiological processes in invertebrates. Nevertheless, it is not clear whether these invertebrate counterparts of norepinephrine are important regulators of metabolic and life history traits. This study shows that flies (Drosophila melanogaster) lacking OA are more resistant to starvation, while their overall life span is substantially reduced compared with control flies. In addition, these animals have increased body fat deposits, reduced physical activity and a reduced metabolic resting rate. Increasing the release of OA from internal stores induced the opposite effects. Flies devoid of both OA and TA had normal body fat and metabolic rates, suggesting that OA and TA act antagonistically. Moreover, OA-deficient flies show increased insulin release rates. It was inferred that the OA-mediated control of insulin release accounts for a substantial proportion of the alterations observed in these flies. Apparently, OA levels control the balance between thrifty and expenditure metabolic modes. Thus, changes in OA levels in response to external and internal signals orchestrate behaviour and metabolic processes to meet physiological needs. Moreover, chronic deregulation of the corresponding signalling systems in humans may be associated with metabolic disorders, such as obesity or diabetes (Li, 2016).

    Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila

    Starvation induces sustained increase in locomotion, which facilitates food localization and acquisition and hence composes an important aspect of food-seeking behavior. This study investigated how nutritional states modulate starvation-induced hyperactivity in adult Drosophila. The receptor of adipokinetic hormone (AKHR), the insect analog of glucagon, is required for starvation-induced hyperactivity. AKHR is expressed in a small group of octopaminergic neurons in the brain. Silencing AKHR+ neurons and blocking octopamine signaling in these neurons eliminates starvation-induced hyperactivity, whereas activation of these neurons accelerates the onset of hyperactivity upon starvation. Neither AKHR nor AKHR+ neurons are involved in increased food consumption upon starvation, suggesting that starvation-induced hyperactivity and food consumption are independently regulated. Single cell analysis of AKHR+ neurons identified the co-expression of Drosophila insulin-like receptor (dInR), which imposes suppressive effect on starvation-induced hyperactivity. Therefore, insulin and glucagon signaling exert opposite effects on starvation-induced hyperactivity via a common neural target in Drosophila (Yu, 2016).

    Food seeking and food consumption are essential for the acquisition of food sources, and hence survival, growth, and reproduction of animal species. Starvation influences food-seeking behavior via both modulating the perception of food cues as well as enhancing flies' locomotor activity. Accumulated evidence has suggested that starvation modulates the activity of ORNs via multiple neural and hormonal cues, which in turn facilitates odor driven food search and food consumption. Similarly, starvation also modulates the perception of food taste via the relative sensitivity of appetitive sweet-sensing and aversive bitter-sensing GRNs,which may in turn increase the attractiveness of food taste. However, how starvation increases the locomotor activity of flies remains largely uncharacterized (Yu, 2016).

    Consistent with previous reports, this study has shown that starved fruit flies exhibit sustained increase in their locomotor activity, which can be suppressed by food consumption induced by both nutritive and non-nutritive food cues. The present study has shown that a small group of neurons located in the subesophageal zone (SEZ) region of the fly brain are both necessary and sufficient for starvation induced hyperactivity. These neurons sense the changes in flies' internal nutritional states by directly responding to two sets of hormones, AKH and DILPs, and modulate locomotor activity in response. Single cell analysis has identified that these AKHR+dInR+ neurons are octopaminergic, which offers an entry point to trace the downstream neural circuitry that regulates starvation-induced hyperactivity. For example, there are seven candidate octopamine receptors in fruit flies and it would be of interest to investigate whether any of these receptors and the receptor-expressing neurons are involved in locomotor regulation upon starvation (Yu, 2016).

    AKH and DILPs are two sets of functionally counteracting hormones in fruit flies. As its mammalian analog glucagon, the reduction in circulating sugars induces the release of AKH, which in turn mobilizes fat storage and provides energy supply for flies. In contrast, DILPs, the insect analog of mammalian insulin, function as satiety hormones. Dietary nutrient induces the release of DILPs into the hemolymph, which in turn promotes protein synthesis, body growth, and other anabolic processes. This study has shown that these two hormonal signaling systems exert opposite effects on starvation-induced hyperactivity via a small group of AKHR+InR+ octopaminergic neurons. These results suggest that these AKHR+dInR+ neurons can integrate the inputs from the two hormonal signaling systems representing hunger and satiety at the same time, and modulate flies' locomotor activity. This elegant yet concise design allows these neurons to be responsive to rapid changes in the internal nutritional states as well as food availability. Furthermore, it is possible that besides hunger and satiety, other physiological states such as wakefulness, stress, and emotions also influence flies' locomotor activity. Notably, single cell analysis has shown that these AKHR+dInR+ neurons also sparsely express other neuropeptide receptors, suggesting that at least small portions of these neurons may also receive input from other neuropeptidergic systems (Yu, 2016).

    Starved animals exhibited increased locomotion and food consumption, the transition of which relies on the detection of food cues. But whether these two behaviors are interdependently or independently regulated remains unclear. This study has shown that these two behaviors are dissociable from each other in fruit flies. On the one hand, although AKHR+ neurons exert robust modulatory effect on starvation-induced hyperactivity, these neurons are neither necessary nor sufficient for starvation-induced food consumption. On the other hand, the regulation of food consumption is independent of starvation-induced hyperactivity as well. Previous studies have shown that a small subset of GABAergic neurons in the fly brain regulates food consumption but exerts no effect on 10 starvation-induced hyperactivity (Pool, 2014). In addition, several neuropeptides are known to regulate food consumption, such as Hugin, NPF, sNPF, Leucokinin, and AstA. However this study found in an RNAi screen that the receptors of these neuropeptides were not involved in the regulation of starvation-induced hyperactivity. Taken together, it is likely that starvation-induced hyperactivity and food consumption are independently regulated by different sets of hormonal cues, and that AKHR+ neurons are only involved in the former but not the latter. These results may shed light on the regulation of food intake in mammals, especially whether starvation-induced hyperactivity and food consumption are also independently regulated by different sets of hormones and distinct neural circuitry in mammals (Yu, 2016).

    PPL2ab neurons restore sexual responses in aged Drosophila males through dopamine

    Male sexual desire typically declines with ageing. However, understanding of the neurobiological basis for this phenomenon is limited by knowledge of the brain circuitry and neuronal pathways controlling male sexual desire. A number of studies across species suggest that dopamine (DA) affects sexual desire. This study used genetic tools and behavioural assays to identify a novel subset of DA neurons that regulate age-associated male courtship activity in Drosophila. Increasing DA levels in a subset of cells in the PPL2ab neuronal cluster is necessary and sufficient for increased sustained courtship in both young and aged male flies. These results indicate that preventing the age-related decline in DA levels in PPL2ab neurons alleviates diminished courtship behaviours in male Drosophila. These results may provide the foundation for deciphering the circuitry involved in sexual motivation in the male Drosophila brain (Kuo, 2015).

    A decision underlies phototaxis in an insect

    Like a moth into the flame-phototaxis is an iconic example for innate preferences. Such preferences probably reflect evolutionary adaptations to predictable situations and have traditionally been conceptualized as hard-wired stimulus-response links. Perhaps for that reason, the century-old discovery of flexibility in Drosophila phototaxis has received little attention. This study reports that across several different behavioural tests, light/dark preference tested in walking is dependent on various aspects of flight. If flying ability is temporarily compromised, walking photopreference reverses concomitantly. Neuronal activity in circuits expressing dopamine and octopamine, respectively, plays a differential role in photopreference, suggesting a potential involvement of these biogenic amines in this case of behavioural flexibility. It is concluded that flies monitor their ability to fly, and that flying ability exerts a fundamental effect on action selection in Drosophila. This work suggests that even behaviours which appear simple and hard-wired comprise a value-driven decision-making stage, negotiating the external situation with the animal's internal state, before an action is selected (Gorostiza, 2016).

    Interestingly, experiments described by McEwen in 1918 and Benzer in 1967 demonstrated that wing defects affect phototaxis also in walking flies. These early works showed that flies with clipped wings did not display the phototactic response to light, whereas cutting the wings from mutants with deformed wings did not decrease their already low response to light any further. The fact that manipulating an unrelated organ, such as wings, affects phototaxis contradicts the assumed hard-wired organization of this behaviour, suggesting that it may not be a simple matter of stimulus and rigid, innate response, but that it contains at least a certain element of flexibility. This work systematically addressed the factors involved in this behavioural flexibility and begin to explore the neurobiological mechanisms behind it (Gorostiza, 2016).

    McEwen's discovery is of interest because of its implications for the supposed rigidity of simple behaviours. The findings of McEwen and Benzer that wing manipulation leads to a decrease in Drosophila phototaxis were reproduced in this study. Slightly altering the conditions of Benzer's countercurrent paradigm (BCP) and comparing performance between two additional experiments, this study found that the decrease in phototaxis is not due to hypoactivity of wing-manipulated flies, but to a more general change in the flies' assessment of their environment. Evidence was discovered that the BCP is just one of several experiments that can measure a fly's general photopreference. Manipulating the wings modulated this preference in all of the selected experiments such that compromised wing utility yielded a decreased preference for brightness (bright stimuli) and an increased preference for darkness (dark stimuli) across the experiments chosen. However, of these experiments, only the BCP can be argued to test phototaxis proper. In Buridan's paradigm, the flies walk between two unreachable black stripes; and in the T-maze, the flies choose between a dark tube and a bright one where the light is coming from an angle perpendicular to their trajectory. Neither of the two paradigms is testing taxis to nor away from a light source. Interestingly, in pilot experiments, this study tested phototaxis in different variations of the T-maze with various LEDs placed at the end of one of two opaque tubes, and only found a reduction of phototaxis and never negative phototaxis. In fact, in these pilot experiments, every possible difference was observed between flying and manipulated flies. In the end, the experimental design was chosen that yielded positive and negative scores, respectively, in wild-type Berlin (WTB) flies purely for practical reasons. Other wild-type strains, such as some Canton S substrains, do not show a negative photopreference in the T-maze after wing clipping. Taken together, these lines of evidence strongly suggest that photopreference in Drosophila is a strain-specific continuum where experimental design assigns more or less arbitrary values along the spectrum. In some special cases, this photopreference manifests itself as phototaxis. If that were the case, phototaxis would constitute an example of a class of experiments not entailing a class of behaviours (Gorostiza, 2016).

    This insight entails that manipulations of different aspects of flight ought to affect this continuum in different ways. Complete loss of flight ought to have more severe effects than manipulations affecting merely individual aspects of flight behaviour, such as wing beat amplitude/frequency (i.e., lift/thrust), torque, flight initiation, flight maintenance, proprioception or motion/wind-speed sensation. This study found some evidence to support this expectation. For instance, clipping only the tips of the wings does not eliminate flight, but affects torque as well as lift/thrust. Flies with the tips of their wings cut behave indifferently in the T-maze and do not avoid the bright tube. Flies without antennae are reluctant to fly and have lost their main sense of air speed detection, but they are still able to fly. Also these flies do not become light averse in the T-maze after the manipulation, but indifferent. Only clipping the wings in these flies abolishes their flight capabilities completely and yields negative scores. Flies with removed gyroscopic halteres, on the other hand, are severely affected in their detection of rotations and usually do not fly, despite being able to still beat their wings and control flight direction using vision alone in stationary flight. These flies avoid the bright arm of the T-maze. Finally, injuries to flight-unrelated parts of the fly's body did not affect photopreference ruling out the preference of darkness being a direct escape response due to bodily harm. Further research is required to establish a quantitative link between the many different aspects of flight behaviour and their relation to photopreference (Gorostiza, 2016).

    Taken together, these experiments so far demonstrate that: (1) the physical state of the wings with regard to their shape, form or degree of intactness influences photopreference; (2) the capability to not just move the wings, but specifically to move them in a way that would support flight also influences the flies' photopreference; and (3) the state of sensory organs related to flight such as antennae or halteres also exerts such an influence, while non-flight-related sensory deprivation shows no such consequences. This multitude of flight-related aspects extends the concept of flying ability beyond mere wing utility: manipulating seemingly any aspect of the entire sensorimotor complex of flight will affect photopreference, and do so reversibly). As it appears that any aspect of flight, sensory or motor, is acutely linked to photopreference, it is straightforward to subsume all of these aspects under the term 'flying ability', emphasizing that flying ability encompasses several more factors in addition to wing utility. The observation that each fly, when it is freshly eclosed from the pupal case and the wings are not yet expanded, goes through a phase of reduced phototaxis that extends beyond wing expansion until the stage when its wings render it capable of flying lends immediate ethological value to a neuronal mechanism linking flying ability with photopreference (Gorostiza, 2016).

    One possible explanation of how the link between flying ability and photopreference may be established mechanistically is via a process reminiscent of learning: at one time point, the flies register a sensory or motor deficit in their flight system and at a later time point they use this experience when making a decision that does not involve flying. Once flying ability is restored, the same choice situation is solved with a different decision again in the absence of flight behaviour. How the flies accomplish this learning task, if indeed learning is involved, is yet unknown, but it is tentatively concluded that it is unlikely that any of the known learning pathways or areas involved in different forms of learning play more than a contributing role. While the molecular learning mechanism remains unidentified, the process appears to be (near) instantaneous. Even though it is not possible to rule out that an unknown learning mechanism exists which is unaccounted for in the screen, it is concluded that at least none of the known learning mechanisms suffices to explain the complete effect size of the shift in photopreference. These results corroborate the findings above, that the switch is instantaneous and does not require thorough training or learning from repeated attempts to fly, let alone flight bouts. They do not rule out smaller contributions due to these known learning processes or an unknown, fast, episodic learning process. It is also possible that the flies constantly monitor their flying ability and hence do not have to remember their flight status. Despite these ambiguities, this study has been able to elucidate some of the underlying neurobiological mechanisms. Much as in other forms of insect learning and valuation, neurons expressing the biogenic amine neuromodulators OA and DA appear to have opposite functions in the modulation of photopreference (Gorostiza, 2016).

    Although both DA and OA play some role in different aspects of flight behaviour, these cannot explain the results. In general, the biogenic amine neuron manipulated flies escape their vial via flight if granted the opportunity. Thus, flight is not abolished in any of the transgenic lines affecting OA, TA or DA neurons. However, there may be more subtle deficits in less readily perceived aspects of flight. Experiments performed with mutant flies lacking OA demonstrated that OA is necessary for initiation and maintenance of flight. However, in the paradigm used in this study, silencing OA/TA neurons promoted approaching light, the opposite effect of what would be expected for a flightless fly. Activating these OA/TA neurons, however, rendered the flies indifferent in the T-maze. OA/TA appear to be involved in flight initiation and maintenance via opponent processes. Transient activation of OA/TA neurons may lead to a subtle alteration of flight performance and reduce photopreference in these flies. Similarly, it has been shown that altering the development of specific DA neurons results in flight deficits (reduction of flight time or loss of flight, depending on the treatment. the manipulations lasted for approximately 30 min during adulthood, ruling out such developmental defects. Work in the laboratory of Gaiti Hasan has also found that silencing of three identified TH-positive interneurons for several days in the adult animal compromises flight to some extent (wing coordination defects during flight initiation and cessation) (Sadaf, 2015). The much shorter manipulation of the current study does not lead to any readily observable flight defect. However, one need not discuss whether or not the aminergic manipulations may have had subtle effects on some aspects of flight behaviour, as these flies can be compared to the wing-clipped siblings with which they were tested simultaneously (i.e., the flies with the maximum shift in photopreference due to completely abolished flight). Comparing the intact DA-inactivated flies and OA/TA-activated flies with their respective wingless siblings (reveals that the choice indices of the pairs of groups become essentially indistinguishable at the restrictive temperature. In other words, intact flies where DA neurons have been inactivated or OA/TA neurons have been activated behave as if their wings had been clipped and their flight capabilities abolished completely, despite them being capable of at least some aspects of flight. Hence, even if there were some contribution of some aspect of flight behaviour being subtly affected by manipulating these aminergic neurons, there is a contribution of activity in these neurons that goes beyond these hypothetical flight deficits. Therefore, it is concluded that neither the OA/TA nor the DA effects can be explained only by subtle defects in one or the other aspect of flight behaviour in the manipulated flies (Gorostiza, 2016).

    The precise neurobiological consequences of manipulating OA/TA and DA neurons, respectively, are less certain, however. The two driver lines (th-GAL4 and tdc2-GAL4) only imperfectly mimicking the expression patterns of the genes from which they were derived. The effectors, moreover, only manipulated the activity of the labelled neurons. One manipulation (shiTS) prevents vesicle recycling and probably affects different vesicle pools differentially, depending on their respective release probabilities and recycling rates. The other effector (TrpA1) depolarizes neurons. It is commonly not known if the labelled neurons may not be co-releasing several different transmitters and/or modulators in the case of supra-threshold depolarization. Hence, without further research, the involvement can be stated only of the labelled neurons, which as populations are likely to be distinct mainly by containing either DA or OA/TA, respectively. Whether it is indeed the release of these biogenic amines or rather the (co-)release of yet unknown factors in these neuronal populations remains to be discovered. Further research will also elucidate the exact relationship between the activities of these two neuronal populations and whether/how it shifts after manipulations of flying ability (Gorostiza, 2016).

    In conclusion, the current findings provide further evidence that even innate preferences, such as those expressed in classic phototaxis experiments, are not completely hard-wired, but depend on the animal's state and presumably other factors, much like in the more complex behaviours previously studied. This endows the animal with the possibility to decide, for example, when it is better to move towards the light or hide in the shadows. Moreover, the fact that flies adapt their photopreference in accordance with their flying ability raises the tantalizing possibility that flies may have the cognitive tools required to evaluate the capability to perform an action and to let that evaluation impact other actions - an observation reminiscent of meta-cognition (Gorostiza, 2016).

    Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain

    The morphology and physiology of neurons are directed by developmental decisions made within their lines of descent from single stem cells. Distinct stem cells may produce neurons having shared properties that define their cell class, such as the type of secreted neurotransmitter. This study developed the transgenic cell class-lineage intersection (CLIn) system to assign cells of a particular class to specific lineages within the Drosophila brain. CLIn also enables birth-order analysis and genetic manipulation of particular cell classes arising from particular lineages. The power of CLIn was demonstrated in the context of the eight central brain type II lineages, which produce highly diverse progeny through intermediate neural progenitors. 18 dopaminergic neurons from three distinct clusters were mapped to six type II lineages that show lineage-characteristic neurite trajectories. In addition, morphologically distinct dopaminergic neurons are produced within a given lineage, and they arise in an invariant sequence. Type II lineages that produce doublesex- and fruitless-expressing neurons were identified, and whether female-specific apoptosis in these lineages accounts for the lower number of these neurons in the female brain was examined. Blocking apoptosis in these lineages results in more cells in both sexes with males still carrying more cells than females. This argues that sex-specific stem cell fate together with differential progeny apoptosis contribute to the final sexual dimorphism (Ren, 2016).

    The relationship between neuron classes and lineages is complex in the Drosophila brain, where analogous neurons of a given class may arise from distinct lineages and a single lineage can yield multiple neuron classes. Therefore, a method was developed that would enable mapping and and analysis of neuron classes with respect to lineage identity using intersectional transgenic strategies. Specifically, the neuron class of interest expresses the GAL4 transcriptional activator from a class-specific transgene, while the lineage(s) of interest expresses the KD recombinase from a lineage-specific transgene. The KD recombinase activity triggers production of another recombinase, Cre, under the control of the deadpan (dpn) promoter, which is active in all NBs. Cre recombinase activity then triggers the simultaneous production of the LexA::p65 transcriptional activator and loss of the GAL4 inhibitor, GAL80, in all subsequently born progeny within the lineage(s). The LexA::p65 activates reporter-A expression within lineages of interest via lexAop. Because all other neurons outside lineage(s) of interest express GAL80, GAL4 is only active in neurons of the LexA::p65-expressing lineage(s) and thus can positively mark these neurons by activating expression of a reporter-B under UAS control. One can therefore subdivide any complex set of neurons that express a class-specific GAL4 transgene based on their developmental lineage(s). Consequently, CLIn enables the unambiguous determination of the lineage origins of particular neuron classes, which is essential for understanding the development and organization of the Drosophila brain (Ren, 2016).

    The CLIn system unambiguously establishes the correspondence between cell classes and their lineage origins and enables the subdivision of a given neuronal class among certain NB lineages. It also allows interrogation of serially derived neuronal diversity. One can therefore map individual neurons of a given class with respect to their lineage and temporal origins in an effort to unravel the intricate neuron class-lineage relationships in the brain (Ren, 2016).

    Revealing diverse cell classes of a lineage, by carefully choosing different GAL4 drivers that each distinguish a particular cell class, will allow better characterization of progeny heterogeneity within a lineage. It is therefore possible to explore how cellular diversity is generated during development. For example, it will be interesting to determine whether a specific cell class develops from one fixed temporal window. Moreover, comparing the cell-class diversity of different lineages will provide insight into the developmental heterogeneity of stem cells (Ren, 2016).

    Conversely, for cell classes that originate from multiple lineages, CLIn analysis reveals the distribution of a cell class among different lineages. Vertebrate studies found that neurons of the same lineage origin, compared to neurons of the same class but different lineage origins, are more likely structurally connected via gap junction and have similar network functions. In Drosophila, lineage has been shown to be a developmental and a functional unit. Thus, assigning a cell class to different lineages may reveal the particular function of a neuronal subset within a cell class (Ren, 2016).

    Moreover, the CLIn system permits incorporations of additional effectors driven by the GAL4-UAS system or the LexA system to manipulate cell class or lineage, respectively. The toolkit of effectors for different purposes is growing rapidly over recent years. Multiple reporter constructs are available to label specific sub-domains of the cell (dendrite, axon, or synapse). Effectors that affect cell viability could eliminate or immortalize specific neurons or glia. Effectors that alter membrane activity can be used to modulate neural activity. In addition, CLIn enables distinguishing gene’s functions in whole lineage including stem and progenitor cells versus only in a subset of lineage progeny by independent gene manipulations via lexAop versus UAS systems (Ren, 2016).

    However, the CLIn system requires further improvement to reach its full potential. In particular, the drivers for targeting various NB subsets remain to be fully characterized. Moreover, their targeting efficiency and specificity could vary individually. Engineering drivers based on genes known to be expressed in defined subsets of embryonic NBs may provide an initial complete set of more reliable NB drivers. An additional challenge for the study of type II lineages is how to selectively target INP sublineages. Via the current dpn enhancer, the frequencies of INP1 sublineages are very low compared with that of NB lineages (Ren, 2016).

    Type II NBs yield supernumerary neurons plus glia, which are expected to be highly diverse in cell classes. CLIn unambiguously assigned various neuronal classes to common type II lineages. In this study, the majority of progeny remained negative for the drivers employed. Revealing the full spectrum of neuronal heterogeneity within type II lineages requires characterization of additional cell-class drivers (Ren, 2016).

    Diverse cell classes could arise from a single INP. Single-cell lineage analysis has shown that one INP can produce multiple morphological classes of neurons most likely pertaining to different functional classes. Temporal mapping by CLIn revealed the birth of both TH-GAL4 and dsxGAL4 neurons in early windows of larval type II lineages. This lends further support to the production of diverse neuronal classes by common INPs. Examining INP clones labeled by CLIn did validate that the first larval-born INP of the DM6 lineage makes one fruGAL4 neuron in addition to two TH-GAL4 neurons (Ren, 2016).

    Per the limited cell-lineage analysis along the NB axis of type II lineages, sibling INPs produce morphologically similar series of neurons that differ in subsets of terminal neurite elaborations. These phenomena indicate expansion of related neurons across sibling INP sublineages. Assuming production of about 50 sibling INPs and in the absence of apoptosis, one would expect composition of 50 cell units for each neuronal class made by one type II NB. Notably, rescuing apoptotic dsx- or fru-expressing neurons throughout lineage development did restore complements of 50 or so cells in several, but not all, type II lineages. However, most type II lineages yield very few, if any, TH-GAL4 neurons. For instance, the DL1 lineage produces two TH-GAL4 neurons that innervate the upper FB layers. Temporal mapping of the DL1 lineage reveals the existence of multiple (n > 3) morphologically distinct INP clones that contain neurons projecting to the FB upper layers, similar to the DL1 TH-GAL4 neurons. Thus, morphologically similar neurons may belong to different functional classes, highlighting the challenges in sorting out neuronal classes and their interrelationships in the brain (Ren, 2016).

    Pioneering studies in C. elegans showed that the acquisition of neurotransmitter identity could be achieved through distinct mechanisms. A shared regulatory signature consisting of three terminal-selector types of transcription factors regulates the terminal identity of all dopaminergic neurons. By contrast, different combinations of terminal selectors act in distinct subsets of glutamatergic neurons to initiate and maintain their glutamatergic identity. In the present study, it was observed that six type II lineages produce 18 dopaminergic neurons but all during early larval neurogenesis. The derivation of TH neurons from multiple neuronal lineages at similar temporal windows argues for their specification by combinations of different lineage-identity genes with common temporal factors (Ren, 2016).

    Previous analysis of fruGAL4 neurons has uncovered 62 discrete MARCM clones in the fly central brain that might arise from an equal number of lineages. Ten clones show dimorphic cell numbers, and 22 clones exhibit dimorphic trajectories. Contrasting the stochastic clonal labeling of only fruGAL4 neurons, CLIn allows determination among a collection of lineages of whether a given lineage yields any fruGAL4 neurons. Based on the additional lineage information, two clones (pIP-j and pIP-h) were attributed as being partial clones of another two full-sized clones (pIP-g and pMP-f). Moreover, a more focused approach reveals sexual dimorphism of fru-expressing neurons in all type II NB lineages (Ren, 2016).

    The presence of many more dsx- or fru-expressing neurons in male than female brains is proposed to result from selective loss of specific neurons in females through apoptosis. However, blocking apoptosis increased dsx- or fru-expressing neurons in both male and female lineages. This is consistent with a previous report showing that sex-independent apoptosis occur widely in type II lineages. Although the number of apoptosis-blocked female neurons was similar, if not identical, to that of the control male neurons, blocking apoptosis unexpectedly increased the number of male dsx- or fru-expressing neurons such that there were more neurons in the apoptosis-blocked male than female lineages. This unmasks the original potential of the male and female NBs to produce different numbers of dsx- or fru-expressing neurons in type II lineages (Ren, 2016).

    Distinct fates have been reported for male and female NBs in the abdominal ganglion of Drosophila CNS. In this study, the male isoform of Dsx, DsxM, promotes additional NB divisions in males relative to females. Similarly, it has been reported that DsxM specifies additional cell divisions in the male, relative to female, central brain NBs that give rise to the pC1 and pC2 clusters. The proliferation of Drosophila intestinal stem cells is also determined by their sexual identity, although this is controlled by genes other than dsx and fru. Consistent with the notion that male and female NBs may possess distinct fates, this study found that male type II lineages contain more neurons committed to express dsx or fru, which possibly results from the greater number of NB divisions in males, as shown in the previous study. Elucidating the underlying molecular mechanisms of sex-specific neuron numbers in the central brain will require additional studies of the sex-dependent production and specification of different dsx- or fru-expressing neurons in the apoptosis-blocked type II NB lineages (Ren, 2016).

    Lineage mapping based on morphology provides limited information about neuronal classes. Given the intricate relationship between neuronal classes and cell lineages, CLIn is needed to resolve the detail even in fly brains where invariant neuronal lineages exist. This is critical for fully understanding how cell lineages guide the formation of variant neural circuits with distinct combinations of neuronal classes and types (Ren, 2016).

    In mammalian brains, extensive neuronal migration obscures the roles of cell lineages in the global organization of neural networks. However, clonally related neurons preferentially make local connections. Moreover, ample evidence exists for the heterogeneity of mammalian neural stem cells and the control of neuronal identity by spatiotemporal patterning of neural progenitors. Untangling of a further sophisticated brain and its development may absolutely require examination of cell lineages and neuronal classes at the same time. Systems like CLIn with its emphasis on the relationship between cell class and lineage potentially aid greatly in the study of mammalian brain development, anatomy, and function (Ren, 2016).

    Electrochemical measurements of optogenetically stimulated quantal amine release from single nerve cell varicosities in Drosophila larvae

    The nerve terminals found in the body wall of Drosophila larvae are readily accessible to experimental manipulation. This study used the light-activated ion channel, channelrhodopsin-2, which is expressed by genetic manipulation in Type II varicosities to study octopamine release in Drosophila. A method was developed to measure neurotransmitter release from exocytosis events at individual varicosities in the Drosophila larval system by amperometry. A microelectrode was placed in a region of the muscle containing a varicosity and held at a potential sufficient to oxidize octopamine and the terminal stimulated by blue light. Optical stimulation of Type II boutons evokes exocytosis of octopamine, which is detected through oxidization at the electrode surface. 22700 +/- 4200 molecules of octopamine were released per vesicle. This system provides a genetically accessible platform to study the regulation of amine release at an intact synapse (Majdi, 2015).

    Four individually identified paired dopamine neurons signal reward in larval Drosophila

    Dopaminergic neurons serve multiple functions, including reinforcement processing during associative learning. It is thus warranted to understand which dopaminergic neurons mediate which function. Larval Drosophila were used, in which only approximately 120 of a total of 10,000 neurons are dopaminergic, as judged by the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine biosynthesis. Dopaminergic neurons mediating reinforcement in insect olfactory learning target the mushroom bodies, a higher-order "cortical" brain region. Four previously undescribed paired neurons, the primary protocerebral anterior medial (pPAM) neurons, were discovered. These neurons are TH positive and subdivide the medial lobe of the mushroom body into four distinct subunits. These pPAM neurons are acutely necessary for odor-sugar reward learning and require intact TH function in this process. However, they are dispensable for aversive learning and innate behavior toward the odors and sugars employed. Optogenetical activation of pPAM neurons is sufficient as a reward. Thus, the pPAM neurons convey a likely dopaminergic reward signal. In contrast, DL1 cluster neurons convey a corresponding punishment signal, suggesting a cellular division of labor to convey dopaminergic reward and punishment signals. On the level of individually identified neurons, this uncovers an organizational principle shared with adult Drosophila and mammals. The numerical simplicity and connectomic tractability of the larval nervous system now offers a prospect for studying circuit principles of dopamine function at unprecedented resolution (Rohwedder, 2016).

    Disruption of dopamine homeostasis has sexually dimorphic effects on senescence characteristics of Drosophila melanogaster

    This study investigated sexually dimorphic effects of disruptions in dopamine (DA) homeostasis and its relationship to senescence using three different Drosophila melanogaster mutants, Catsup (Catsup26) with elevated DA levels, and pale (ple2), Punch (PuZ22) with depleted DA levels. In all genotypes including controls, DA levels were significantly lower in old (45-50-day-old) flies compared with young (3-5-day-old) in both sexes. Interestingly, females have lower DA content than males at young age whereas this difference is not observed in old age, suggesting that males have a larger decline in DA levels with age. Females, in general, are longer lived compared with males in all genotypes except ple2 mutants with depleted DA levels, and females also demonstrate marked age-related decline in circadian locomotor activity compared with males. Old Catsup26 males with elevated DA levels accumulate significantly lower levels of lipid peroxidation product 4-hydroxy 2-nonenal compared with wild type, ple2 and PuZ22 mutant males. A sexually dimorphic response was also observed in the expression levels of key stress and aging associated transcription factors across genotypes with elevated or depleted DA levels. Taken together, these results reveal a novel sexually dimorphic involvement of DA in senescence characteristics of D. melanogaster (Bednářová, 2017).

    Active and passive sexual roles that arise in Drosophila male-male courtship are modulated by dopamine levels in PPL2ab neurons

    The neurology of male sexuality has been poorly studied owing to difficulties in studying brain circuitry in humans. Dopamine (DA) is essential for both physiological and behavioural responses, including the regulation of sexuality. Previous studies have revealed that alterations in DA synthesis in dopaminergic neurons can induce male-male courtship behaviour, while increasing DA levels in the protocerebral posteriolateral dopaminergic cluster neuron 2ab (PPL2ab) may enhance the intensity of male courtship sustainment in Drosophila. This study reports that changes in the ability of the PPL2ab in the central nervous system (CNS) to produce DA strongly impact male-male courtship in D. melanogaster. Intriguingly, the DA-synthesizing abilities of these neurons appear to affect both the courting activities displayed by male flies and the sex appeal of male flies for other male flies. Moreover, the observed male-male courtship is triggered primarily by target motion, yet chemical cues can replace visual input under dark conditions. This is interesting evidence that courtship responses in male individuals are controlled by PPL2ab neurons in the CNS. This study provides insight for subsequent studies focusing on sexual circuit modulation by PPL2ab neurons (Chen, 2017).

    Sleep facilitates memory by blocking dopamine neuron-mediated forgetting

    Early studies from psychology suggest that sleep facilitates memory retention by stopping ongoing retroactive interference caused by mental activity or external sensory stimuli. Neuroscience research with animal models, on the other hand, suggests that sleep facilitates retention by enhancing memory consolidation. Recently, in Drosophila, the ongoing activity of specific dopamine neurons was shown to regulate the forgetting of olfactory memories. This study shows that this ongoing dopaminergic activity is modulated with behavioral state, increasing robustly with locomotor activity and decreasing with rest. Increasing sleep-drive, with either the sleep-promoting agent Gaboxadol or by genetic stimulation of the neural circuit for sleep, decreases ongoing dopaminergic activity, while enhancing memory retention. Conversely, increasing arousal stimulates ongoing dopaminergic activity and accelerates dopaminergic-based forgetting. Therefore, forgetting is regulated by the behavioral state modulation of dopaminergic-based plasticity. These findings integrate psychological and neuroscience research on sleep and forgetting (Berry 2015).

    While some memories are long-lasting, most others fade away and are forgotten. Why we forget, has been an intriguing and central question in psychology and neuroscience for more than a century. Even though forgetting is often thought of as a failure or limitation of the brain, recent studies support the view that forgetting is a biologically regulated function of the brain allowing optimal adaptability to an ever-changing environment. In the fruit fly Drosophila, it was recently shown that the very same set of dopamine neurons (DANs) that signal through one receptor to form aversive olfactory memories, also signal through a separate receptor after learning to forget these memories (Berry, 2012). However, it remains unclear whether this dopaminergic forgetting signal is constant and autonomous, or dynamic and regulated (Berry 2015).

    From fruit flies to humans, animals routinely alternate between highly active behavioral states and long states of immobility and quiescence called sleep. Despite the obvious disadvantages an inanimate state conveys to survival, sleep has been proposed to have critically important functions, including in memory and cognition. Since the earliest experimental studies of human memory, sleep shortly after learning has been shown to consistently lead to an increase in retention and thus less forgetting of many forms of memory including declarative and emotional memory in mammals and long-term courtship memory in Drosophila. However, there exists controversy as to how sleep benefits memory retention. Many studies in mammals support the idea that sleep benefits memory retention because it is accompanied by specific mechanisms, such as slow wave sleep, rapid eye movement (REM) sleep, and sharp-wave ripple-based memory replay, that increase memory retention by actively consolidating newly formed memories. Alternatively, it was proposed nearly a century ago and recently revisited, that sleep, or long periods of quiet wakefulness, benefit memory retention by muting experience-driven plasticity and new memory formation, thus reducing retroactive interference-based forgetting. In addition, this state of reduced neuronal activity might then allow consolidation to occur more efficiently, referred to as the "opportunistic consolidation" model. Thus, the essence of how exactly sleep benefits memory retention remains debated (Berry 2015).

    Previous studies have observed that, after promoting the acquisition of olfactory memories, a small set of DANs that innervate the mushroom body (MB) memory center, intriguingly, display synchronized and ongoing Ca2+-based activity after learning that causes the forgetting of early aversive olfactory memories in Drosophila(Berry, 2012). While this activity occurs as reoccurring bursts, it was noticed that the pattern of activity appeared temporally regulated, occurring in bouts. In order to understand how the DAN-based forgetting signal might be regulated, an in vivo imaging assay was developed allowing simultaneous monitoring of a fly's DAN Ca2+ activity, via GCaMP3.0 expression using TH-gal4 and behavior while walking on a ball supported by air. Focus was placed on two regions of the DAN processes that form synaptic connections to the MBs, referred to as neuropils, one that displays ongoing activity and belongs to the MV1 neuron and an adjacent control region belonging to the V1 neuron, which is relatively inactive. Remarkably, a 1-hr simultaneous recording of locomotion and DAN activity revealed that the MV1 neuropil displayed activity resembling the coarse temporal pattern of locomotor behavior. Ball rotation data was used to cluster time points into either behaviorally active or rest states, and MV1 neuropil activity was found to be robustly elevated during active states, whereas the V1 neuropil activity remained low in both states, but had a slight decrease during active states. Furthermore, the MV1 neuropil Ca2+ signal was strongly correlated with ball rotation, particularly in the lower frequency domains (frequency < 0.002 Hz, or ~1 cycle every 8 min or more, timescales consistent with that of locomotor bout structure). Finally, DAN activity was examined during stable transitions into and out of behaviorally active states ( by aligning transition segments of recordings across all animals. Interestingly, MV1 DAN activity robustly increased upon transition into active states, while, conversely, dropped during rest states. V1 activity remained low and was not significantly regulated with behavioral transitions. Together, these data, along with observations of synchronized activity between MV1 and another DAN, MP1, indicate that the ongoing activity from specific sets of DAN involved in forgetting, including MV1, is regulated with the behavioral state of the animal (Berry 2015).

    Given the strong correlation between DAN activity and locomotor activity, tests were performed to see whether DAN activation might promote locomotor activity, that is, whether DAN activity is upstream of locomotor circuits. Two prior studies found no role for these MB innervating DANs in regulating locomotor activity. When the synaptic output from these DANs was blocked with restricted expression in MV1, MP1, and V1 DANs to drive temperature-sensitiveUAS-shits1function, no decreased locomotor activity was seen between temperatures, although the experimental genotype exhibited less activity at high temperature compared to one but not both control genotypes. It was also noted from imaging experiments that locomotor activity occasionally occurs while the MV1 neuron is not active, thus further supporting that locomotor behavior does not require c150-gal4>DAN output. Furthermore, stimulation of these neurons, using UAS-trpA1, did not produce genotype specific and robust increases in locomotor activity. But similar to the blocking experiments, high temperature increased the locomotor activity of the two control genotypes (UAS-trpA1 and c150-gal4 alone). These data indicate that c150-gal4 DAN output is neither necessary nor sufficient to acutely drive locomotor activity. It is therefore concluded that the ongoing signal in MV1 is either downstream of locomotor behavior itself, or is regulated in parallel, by other brain areas that promote arousal and locomotor activity (Berry 2015).

    Given that the ongoing activity in MV1 was highest during behaviorally active states, the hypothesis was tested that reducing behavioral activity with increased sleep drive would reduce this ongoing activity. The GABAA agonist, Gaboxadol (or THIP), has been shown to specifically promote deep non-REM sleep in humans, while leaving REM sleep intact; sleep characteristics similar to those occurring during normal homeostatic regulation of sleep. Recently, it was shown that Gaboxadol also induces sleep in Drosophila. To confirm this, attempts were made to induce sleep in Drosophila by feeding flies various doses of Gaboxadol. Shortly after Gaboxadol feeding, long periods of quiescence, (>5 min) conventionally defined as 'sleep' in Drosophila, significantly increased during both day and night in a dose-dependent manner. Next, flies were fed Gaboxadol (0.1 mg/ml) for 1 day and then removed the drug to test whether these effects were reversible. Once again, Gaboxadol treatment increased sleep, occurring as bouts with increased duration, but remarkably, total sleep and bout duration actually decreased after drug removal compared to control fed flies. These data indicate that less sleep is needed in flies given Gaboxadol the prior day, suggestive of a homeostatic response. Finally, to test the arousability of flies fed Gaboxadol, a single mechanical stimulus was delivered every hour for 1 day followed by a day of drug treatment. Interestingly, the average-evoked activity, post-stimulus was significantly reduced with increasing Gaboxadol dosage. These data suggest that having Gaboxadol onboard increases arousal thresholds. Altogether, these data indicate that Gaboxadol, similar to effects on mammals, induces bona fide sleep in Drosophila, with hallmark characteristics that include reversible quiescence, homeostatic regulation, and increased arousal thresholds (Berry 2015).

    In order to observe the effects of Gaboxadol on DAN activity, varying concentrations of this sleep agent were perfused across the brain while performing in vivo imaging of MV1 activity and fly body movement was monitored in a recording chamber. Like walking on the ball, ongoing MV1 activity was also regulated with behavioral state in this assay, increasing during bouts of body movement. Remarkably, Gaboxadol perfusion rapidly and robustly attenuated both fly movement and MV1 activity at 0.01 and 0.1 mg/ml. Furthermore, it was found that the quiescent behavioral state and reduced MV1 activity was fully reversible with wash out, thus eliminating pharmacological-induced damage as a cause of decreased physical and DAN activity (Berry 2015).

    To rule out non-specific effects of Gaboxadol and extend these results, sleep drive was increased sharply by thermogenetic stimulation of the sleep circuit. Recent studies identified a dorsal fan shaped body (dfsb) circuit in the central brain, specifically represented in the R23E10-gal4 line, which acts as the effector arm of the sleep homeostat (Donlea; 2011 and Donlea; 2014). Consistent with these studies, TrpA1-based stimulation of R23E10-gal4-expressing neurons caused a rapid and robust increase in daytime sleep followed by a negative sleep rebound the day after stimulation, confirming the dfsb circuit's role in homeostatic sleep regulation. In order to measure DAN activity in vivo while using the gal4-uas system to modulate the sleep circuit, a TH-lexA line was developed to express GCaMP3.0 in the MV1 and V1 DAN neurons, their associated MB neuropil regions, as well as DAN innervation of the anterior inferior medial protocerebrum (PR), a region also exhibiting ongoing activity like MV1 (Berry, 2012). While simultaneously measuring movement and DAN activity, before ('Pre'), during ('Treat'), and after ('Post') stimulation of dfsb neurons was recorded. As predicted, stimulation of the sleep circuit rapidly decreased fly behavioral activity and was accompanied with a robust decrease in MV1 and PR DAN activity, with no change in the control V1 region. Fly behavioral activity was partially restored and ongoing activity in MV1 and PR completely restored to pre-stimulation levels after stimulation of the sleep circuit was ceased. These results, along with those from Gaboxadol administration, indicate that increased sleep drive dramatically reduces the ongoing activity of DANs involved in forgetting (Berry 2015).

    Since ongoing MV1 activity is decreased with increasing sleep drive, it was hypothesized that acutely and reversibly increasing sleep drive specifically after learning would reduce DAN-mediated forgetting. To test this, sleep was modulated with Gaboxadol after aversive olfactory conditioning, where populations of flies learn to associate an odor with electric shock. Memory to this association is then tested in a T-maze, giving flies the choice between the trained odor and an unconditioned odor. Since memory from this kind of training decays quickly after training, attempts were made to increase the rate of Gaboxadol consumption and thus the rate of sleep onset, by increasing the hunger of flies via starvation prior to feeding. As was observed previously, flies fed Gaboxadol experienced more sleep than controls, and a 16-hr starvation period increased this effect. Furthermore, it was found that flies removed from Gaboxadol food 1 hr after learning partially returned to control sleep and activity levels by the sixth hour and completely by the eighth hour after learning, indicating that these time points were appropriate for testing memory retrieval. This Gaboxadol feeding protocol led to increased sleep during the period of memory retention. Remarkably, it was found that flies forced to sleep with Gaboxadol treatment after learning exhibited enhanced memory retention at both 6 and 8 hr. Similarly, sleep circuit stimulation after conditioning also rapidly and reversibly induced sleep and enhanced both 3- and 6-hr memory retention. Importantly, simultaneous stimulation of the dfsb sleep circuit and c150-gal4 DANs also led to strong sleep induction. Memory retention, in contrast, was markedly decreased, similar to that observed with stimulation of DANs alone. Therefore, sleep, after learning, loses its protective qualities when DAN signaling is artificially potentiated. This circuit level epistasis experiment indicates that DAN-mediated forgetting is downstream of sleep networks. Together, the data indicate that increased sleep and reduced arousal after learning reduces DAN-mediated forgetting of aversive olfactory memories (Berry 2015).

    If the DANs innervating the MB memory center are downstream of arousal circuitry, then they should respond to arousing stimuli. In fact, these neurons have already been shown to respond to many salient stimuli, including odors and electric shock and temperature changes. Since mechanical stimuli have been extensively used to arouse flies for sleep deprivation, airpuffs were delivered to the fly using a protocol shown to induce arousal in flies, while simultaneously recording fly movement and DAN activity. It was found that the DAN processes in all three areas (MV1, PR, V1) of the mushroom body neuropil exhibited robust responses to each airpuff. However, MV1 responsiveness was maintained across stimuli while the other regions showed attenuated responsiveness across stimuli. Importantly, both fly movement and ongoing MV1 activity continued at an elevated level just after stimulation, indicating a stimulus-induced elevation in arousal and MV1 DAN activity (Berry 2015).

    Next, it was reasoned that increasing the arousal after learning would accelerate DAN-mediated forgetting. To test this, a population arousal device was developed that allowed delivery of a mechanical stimuli (2-s stimulus every 1 min over 80 min) to flies in population vials after aversive olfactory learning. It was found that mechanical stimuli delivered for the first 80 min after learning significantly aroused populations of flies, leading to an overall increase in activity between each stimulus, with activity levels dropping back to control levels after treatment. Importantly, mechanical stimulation after learning caused a robust decrease in 3-hr memory for wild-type Canton-S flies. However, acquisition and immediate memory retrieval were not disrupted by prior mechanical stimulation, indicating that the stimuli must be delivered after learning to observe its disruptive effects. Remarkably, it was found that blocking neuronal output of c150-gal4DANs specifically during mechanical stimulation blocked the forgetting induced by this treatment. Therefore, these data indicate that increasing arousal after learning accelerates DAN-mediated forgetting (Berry 2015).

    The following conclusions are made from the data. First, after learning, the ongoing DA forgetting signal is not constant but instead is regulated with behavioral state. Thus, the forgetting signal does not chronically remove memories at a constant rate. Second, the ongoing forgetting signal is coupled directly to the arousal level of the animal, being suppressed with low levels of arousal such as with the state of sleep and being enhanced by activation of sensory pathways. As a result, forgetting decreases when flies rest or sleep and increases when flies are aroused by external stimuli (Berry 2015).

    DA is known to regulate various types of plasticity in mammals. In flies, DA has been shown to elicit presynaptic plasticity within the Kenyon cells of the MB memory center proposed to underlie learning. Additionally, it was previously found that DA after learning regulates forgetting (Berry, 2012), thus implicating a DA-based plasticity mechanism that weakens memories. Synthesizing these previous observations with the current data, it is proposed that the behavioral state-coupled DA signal, discovered in this study, regulates the plasticity of the memory system, making it malleable for memory updating so that memories of current events can be formed and old, unused memories can be forgotten. While it was found previously that different DA receptors underlie learning and forgetting, more work remains to distinguish the molecular cascades involved and the cellular events that underlie these forms of behavioral plasticity (Berry 2015).

    These findings add compelling mechanistic evidence to support the model that sleep, which begins with and is accompanied by inactivity or rest, benefits newly formed labile memories by reducing the level of plasticity induced by behavioral activity. Furthermore, as sleep progresses and arousal thresholds increase, DANs become less reactive to stimuli. Thus, the molecular/cellular model is congruent with early psychological models of sleep benefitting memory by muting the retroactive interference that causes forgetting. Nevertheless, the data do not eliminate the possibility that sleep-specific mechanisms exist that enhance memory consolidation, as often proposed from studies with mammalian systems. Mechanistically, the effects of sleep on memory consolidation and forgetting may operate in parallel and independently of one another or more intriguingly; they may operate in serial in a dependent fashion, with reduced forgetting being a prerequisite for sleep-facilitated consolidation, similar to the 'opportunistic consolidation' model proposed by Mednick, 2011 (Berry 2015).

    This study has observed that multiple DANs produce the ongoing DA signal, synchronized across the MB memory center and protocerebrum, that leads to forgetting of olfactory memories. It remains to be determined if this network activity is but one segment of a larger and more diffuse DA network that operates on memory types other than olfactory; whether there exist multiple, independent forgetting networks regulated by arousal levels; and whether forgetting of non-olfactory memories occurs through DA-based mechanisms or involves other neuromodulatory transmitters (Berry 2015).

    Control of sleep by dopaminergic inputs to the Drosophila mushroom body

    The Drosophila mushroom body (MB) is an associative learning network that is important for the control of sleep. Particular intrinsic MB Kenyon cell (KC) classes have been identified that regulate sleep through synaptic activation of particular MB output neurons (MBONs) whose axons convey sleep control signals out of the MB to downstream target regions. Specifically, it was found that sleep-promoting KCs increase sleep by preferentially activating cholinergic sleep-promoting MBONs, while wake-promoting KCs decrease sleep by preferentially activating glutamatergic wake-promoting MBONs. By using a combination of genetic and physiological approaches to identify wake-promoting dopaminergic neurons (DANs) that innervate the MB, it was shown that they activate wake-promoting MBONs. These studies reveal a dopaminergic sleep control mechanism that likely operates by modulation of KC-MBON microcircuits (Sitaraman, 2015b).

    This study used a combination of sophisticated cell-specific genetic manipulations with behavioral sleep analysis and optical electrophysiology to identify compartment-specific wake-promoting MB DANs that activate wake-promoting microcircuits. Previous studies have implicated DANs innervating the central complex (CX) - a brain region involved in locomotor control - in regulating sleep, and other non-dopamingeric CX neurons have been implicated in homeostatic control of sleep. In addition, it has recently been shown that manipulations of dopamine signaling in the MB alter sleep, although the precise DANs involved remains unclear. This study has now identified specific wake-promoting MB DANs and shown that they innervate lobe compartments also innervated by wake-promoting KCs and MBONs. Importantly, this study has also shown that dopamine secretion by DANs innervating a particular MB lobe compartment acts through D1 subtype receptors to activate the wake-promoting microcircuit specific to that compartment to a much greater extent than it activates the sleep-promoting microcircuit residing in different compartments. This provides direct physiological evidence for compartment-specific dopamine signaling in the regulation of sleep by the MB, and is consistent with a previous study in the context of learning and memory (Boto, 2014). Future studies are required to determine additional cellular and molecular details of how dopamine signals modulate sleep-regulating microcircuits (Sitaraman, 2015b).

    On the basis of recently published studies of MB control of sleep and the results presented in this study, a unified mechanistic model is proposed for homeostatic control of sleep by excitatory microcircuits in the Drosophila MB. Wake-promoting MBON-γ5β'2a/β'2mp/β'2mp_bilateral and sleep-promoting γ2α'1 each receive anatomical inputs from both wake-promoting γm and α'/β' KCs KCs and sleep-promoting γd KCs. However, segregation of sleep control information into separate microcircuits is enforced by greater synaptic weights between γ and γm and α'/β' KCs and MBON-γ5β'2a/β'2mp/β'2mp_bilateral, and between γm and α/β' KCs and MBON-γ5β'2a/β'2mp/β'2mp_bilateral, and between γd KCs and MBON-γ2α'1 (Sitaraman, 2015a). Thus it is hypothesize that compartment-specific dopamine signals from MB DANs could potentially determine these differences in synaptic weight. Future studies will test this hypothesis (Sitaraman, 2015b).

    Interestingly, other fly behaviors have recently been found to be regulated by sleep-controlling compartment-specific MB microcircuits. For example, the integration of food odor to suppress innate avoidance of CO2 is mediated by MBON-γ5β'2a/β'2mp/β'2mp_bilateral and PAM DANs that innervate the β'2 compartment (Lewis, 2015). Optogenetic activation experiments reveal that wake-promoting γ5β'2a/β'2mp/β'2mp_bilateral mediates innate avoidance, while MBON-γ2α'1 mediates attraction. However, thermogenetic inactivation studies reveal that both MBON-γ5β'2a/β'2mp/β'2mp_bilateral and MBON-γ2α'1 are important for various forms of associative memory formation. These diverse waking behaviors that involve the activity of sleep-regulating neurons raises the interesting question whether such roles are independent, or causally linked, which future studies can address (Sitaraman, 2015b).

    Importantly, this study has provided for the first time a cellular and molecular mechanism for for dopaminergic control of sleep through modulation of an associative network. While dopaminergic projections to cerebral cortex are known to be important for regulating sleep and arousal in mammals, underlying cellular and molecular mechanisms remain poorly understood, although D2 subtype dopamine receptors have been implicated in the control of REM sleep. Because of the possible evolutionary relationship between the MB and vertebrate forebrain associative networks (such as mammalian cerebral cortex), these studies thus provide a framework for the design of analogous experiments in genetically tractable vertebrate model systems such as zebrafish and mice (Sitaraman, 2015b)

    Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain"

    Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. This study shows in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMAT's role in mediating amphetamine action in dopamine neurons, novel genetic, pharmacological and optical approaches were used in Drosophila. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H(+) antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, this study found that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects (Freyberg, 2016).

    Characterization of a novel Drosophila SERT mutant: insights on the contribution of the serotonin neural system to behaviors

    A better comprehension on how different molecular components of the serotonergic system contribute to the adequate regulation of behaviors in animals is essential in the interpretation on how they are involved in neuropsychiatric and pathological disorders. It is possible to study these components in "simpler" animal models including the fly Drosophila melanogaster, given that most of the components of the serotonergic system are conserved between vertebrates and invertebrates. This study has attempted advance understanding on how the serotonin plasma membrane transporter (SERT) contributes to serotonergic neurotransmission and behaviors in Drosophila. A mutant for Drosophila SERT (dSERT) was characterized, and additionally a highly selective serotonin-releasing drug, 4-methylthioamphetamine (4-MTA), was used whose mechanism of action involves the SERT protein. The results show that dSERT mutant animals exhibit an increased survival rate in stress conditions, increased basal motor behavior and decreased levels in an anxiety-related parameter, centrophobism. It was also shown that 4-MTA increases the negative chemotaxis towards a strong aversive odorant, Benzaldehyde. The neurochemical data suggest that this effect is mediated by dSERT and depends on 4-MTA-increased release of serotonin in the fly brain. The in silico data support the idea that these effects are explained by specific interactions between 4-MTA and dSERT. In sum neurochemical, in-silico and behavioral analyses demonstrate the critical importance of the serotonergic system and particularly dSERT functioning in modulating several behaviors in Drosophila (Hidalgo, 2017).

    The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for MDMA translocation and the induction of substrate efflux

    The substituted amphetamine MDMA is a widely used drug of abuse that induces non-exocytotic release of serotonin, dopamine, and norepinephrine through their cognate transporters as well as blocking the reuptake of neurotransmitter by the same transporters. The resulting dramatic increase in volume transmission and signal duration of neurotransmitters leads to psychotropic, stimulant, and entactogenic effects. The mechanism by which amphetamines drive reverse transport of the monoamines remains largely enigmatic. Previous, studies has identified functional differences between the human and Drosophila melanogaster serotonin transporters (hSERT and dSERT, respectively) revealing that MDMA is an effective substrate for hSERT but not dSERT even though serotonin is a potent substrate for both transporters. Chimeric dSERT/hSERT transporters revealed that the molecular components necessary for recognition of MDMA as a substrate was linked to regions of the protein flanking transmembrane domains (TM) V through IX. This study performed species-scanning mutagenesis of hSERT, dSERT and C. elegans SERT (ceSERT) along with biochemical and electrophysiological analysis and identified a single amino acid in TM10 (Glu394, hSERT; Asn484, dSERT, Asp517, ceSERT) that is primarily responsible for the differences in MDMA recognition. The findings reveal that an acidic residue is necessary at this position for MDMA recognition as a substrate and serotonin releaser (Sealover, 2016).

    Modulatory action by the serotonergic system: behavior and neurophysiology in Drosophila melanogaster

    Serotonin modulates various physiological processes and behaviors. This study investigates the role of 5-HT in locomotion and feeding behaviors as well as in modulation of sensory-motor circuits. The 5-HT biosynthesis was dysregulated by feeding Drosophila larvae 5-HT, a 5-HT precursor, or an inhibitor of tryptophan hydroxylase during early stages of development. The effects of feeding fluoxetine, a selective serotonin reuptake inhibitor, during early second instars were also examined. 5-HT receptor subtypes were manipulated using RNA interference mediated knockdown and 5-HT receptor insertional mutations. Moreover, synaptic transmission at 5-HT neurons was blocked or enhanced in both larvae and adult flies. The results demonstrate that disruption of components within the 5-HT system significantly impairs locomotion and feeding behaviors in larvae. Acute activation of 5-HT neurons disrupts normal locomotion activity in adult flies. To determine which 5-HT receptor subtype modulates the evoked sensory-motor activity, pharmacological agents were used. In addition, the activity of 5-HT neurons was enhanced by expressing and activating TrpA1 channels or channelrhodopsin-2 while recording the evoked excitatory postsynaptic potentials (EPSPs) in muscle fibers. 5-HT2 receptor activation mediates a modulatory role in a sensory-motor circuit, and the activation of 5-HT neurons can suppress the neural circuit activity, while fluoxetine can significantly decrease the sensory-motor activity (Majeed, 2016).

    Visual attention in flies-dopamine in the mushroom bodies mediates the after-effect of cueing

    Visual environments may simultaneously comprise stimuli of different significance. Often such stimuli require incompatible responses. Selective visual attention allows an animal to respond exclusively to the stimuli at a certain location in the visual field. In the process of establishing its focus of attention the animal can be influenced by external cues. This study characterized the behavioral properties and neural mechanism of cueing in the fly Drosophila melanogaster. A cue can be attractive, repulsive or ineffective depending upon (e.g.) its visual properties and location in the visual field. Dopamine signaling in the brain is required to maintain the effect of cueing once the cue has disappeared. Raising or lowering dopamine at the synapse abolishes this after-effect. Specifically, dopamine is necessary and sufficient in the αβ-lobes of the mushroom bodies. Evidence is provided for an involvement of the αβposterior Kenyon cells (Koenig, 2016).

    Operation of a homeostatic sleep switch

    In Drosophila, a crucial component of the machinery for sleep homeostasis is a cluster of neurons innervating the dorsal fan-shaped body (dFB) of the central complex. dFB neurons in sleep-deprived flies tend to be electrically active, with high input resistances and long membrane time constants, while neurons in rested flies tend to be electrically silent. This study demonstrates state switching by dFB neurons, identifies dopamine as a neuromodulator that operates the switch, and delineates the switching mechanism. Arousing dopamine causes transient hyperpolarization of dFB neurons within tens of milliseconds and lasting excitability suppression within minutes. Both effects are transduced by Dop1R2 receptors and mediated by potassium conductances. The switch to electrical silence involves the downregulation of voltage-gated A-type currents carried by Shaker and Shab, and the upregulation of voltage-independent leak currents through a two-pore-domain potassium channel that was termed Sandman. Sandman is encoded by the CG8713 gene and translocates to the plasma membrane in response to dopamine. dFB-restricted interference with the expression of Shaker or Sandman decreases or increases sleep, respectively, by slowing the repetitive discharge of dFB neurons in the ON state or blocking their entry into the OFF state. Biophysical changes in a small population of neurons are thus linked to the control of sleep-wake state (Pimentel, 2016).

    Recordings were made from dFB neurons (which were marked by R23E10-GAL4 or R23E10-lexA-driven green fluorescent protein (GFP) expression) while head-fixed flies walked or rested on a spherical treadmill. Because inactivity is a necessary correlate but insufficient proof of sleep, the analysis was restricted to awakening, which is defined as a locomotor bout after >5 min of rest, during which the recorded dFB neuron had been persistently spiking. To deliver wake-promoting signals, the optogenetic actuator CsChrimson was expressed under TH-GAL4 control in the majority of dopaminergic neurons, including the PPL1 and PPM3 clusters, whose fan-shaped body (FB)-projecting members have been implicated in sleep control. Illumination at 630 nm, sustained for 1.5 s to release a bolus of dopamine, effectively stimulated locomotion. dFB neurons paused in successful (but not in unsuccessful) trials, and their membrane potentials dipped by 2-13 mV below the baseline during tonic activity. When flies bearing an undriven CsChrimson transgene were photostimulated, neither physiological nor behavioural changes were apparent. The tight correlation between the suppression of dFB neuron spiking and the initiation of movement might, however, merely mirror a causal dopamine effect elsewhere, as TH-GAL4 labels dopaminergic neurons throughout the brain. Because localized dopamine applications to dFB neuron dendrites similarly caused awakening, this possibility is considered remote (Pimentel, 2016).

    Flies with enhanced dopaminergic transmission exhibit a short-sleeping phenotype that requires the presence of a D1-like receptor in dFB neurons, suggesting that dopamine acts directly on these cells. dFB-restricted RNA interference (RNAi) confirmed this notion and pinpointed Dop1R2 as the responsible receptor, a conclusion reinforced by analysis of the mutant Dop1R2MI08664 allele. Previous evidence that Dop1R1, a receptor not involved in regulating baseline sleep, confers responsiveness to dopamine when expressed in the dFB indicates that either D1-like receptor can fulfill the role normally played by Dop1R2. Loss of Dop1R2 increased sleep during the day and the late hours of the night, by prolonging sleep bouts without affecting their frequency. This sleep pattern is consistent with reduced sensitivity to a dopaminergic arousal signal (Pimentel, 2016).

    To confirm the identity of the effective transmitter, avoid dopamine release outside the dFB, and reduce the transgene load for subsequent experiments, optogenetic manipulations of the dopaminergic system were replaced with pressure ejections of dopamine onto dFB neuron dendrites. Like optogenetically stimulated secretion, focal application of dopamine hyperpolarized the cells and suppressed their spiking. The inhibitory responses could be blocked at several nodes of an intracellular signalling pathway that connects the activation of dopamine receptors to the opening of potassium conductances: by RNAi-mediated knockdown of Dop1R2; by the inclusion in the patch pipette of pertussis toxin (PTX), which inactivates heterotrimeric G proteins of the Gi/o family; and by replacing intracellular potassium with caesium, which obstructs the pores of G-protein-coupled inward-rectifier channels. Elevating the chloride reversal potential above resting potential left the polarity of the responses unchanged, corroborating that potassium conductances mediate the bulk of dopaminergic inhibition (Pimentel, 2016).

    Coupling of Dop1R2 to Gi/o, although documented in a heterologous system, represents a sufficiently unusual transduction mechanism for a predicted D1-like receptor to prompt verification of its behavioural relevance. Like the loss of Dop1R2, temperature-inducible expression of PTX in dFB neurons increased overall sleep time by extending sleep bout length (Pimentel, 2016).

    While a single pulse of dopamine transiently hyperpolarized dFB neurons and inhibited their spiking, prolonged dopamine applications (50 ms pulses at 10 Hz, or 20 Hz optogenetic stimulation, both sustained for 2-10 min) switched the cells from electrical excitability (ON) to quiescence (OFF). The switching process required dopamine as well as Dop1R2, but once the switch had been actuated the cells remained in the OFF state-and flies, awake-without a steady supply of transmitter. Input resistances and membrane time constants dropped to 53.3 ± 1.8 and 24.0 ± 1.3% of their initial values (means ± s.e.m.), and depolarizing currents no longer elicited action potentials (15 out of 15 cells). The biophysical properties of single dFB neurons, recorded in the same individual before and after operating the dopamine switch, varied as widely as those in sleep-deprived and rested flies (Pimentel, 2016).

    Dopamine-induced changes in input resistance and membrane time constant occurred from similar baselines in all genotypes and followed single-exponential kinetics with time constants of 1.07-1.10 min. The speed of conversion points to post-translational modification and/or translocation of ion channels between intracellular pools and the plasma membrane as the underlying mechanism(s). In 7 out of 15 cases, recordings were held long enough to observe the spontaneous recommencement of spiking, which was accompanied by a rise to baseline of input resistance and membrane time constant, after 7-60 min of quiescence (mean ± s.e.m. = 25.86 ± 7.61 min). The temporary suspension of electrical output is thus part of the normal activity cycle of dFB neurons and not a dead end brought on by the experimental conditions (Pimentel, 2016).

    dFB neurons in the ON state expressed two types of potassium current: voltage-dependent A-type (rapidly inactivating) and voltage-independent non-A-type currents. The current-voltage (I-V) relation of iA resembled that of Shaker, the prototypical A-type channel: no current flowed below -50 mV, the approximate voltage threshold of Shaker; above -40 mV, peak currents increased steeply with voltage and inactivated with a time constant of 7.5 ± 2.1 ms (mean ± s.e.m.). Non-A-type currents showed weak outward rectification with a reversal potential of -80 mV, consistent with potassium as the permeant ion, and no inactivation (Pimentel, 2016).

    Switching the neurons OFF changed both types of potassium current. iA diminished by one-third, whereas inon-A nearly quadrupled when quantified between resting potential and spike threshold. The weak rectification of inon-A in the ON state vanished in the OFF state, giving way to the linear I-V relationship of an ideal leak conductance. dFB neurons thus upregulate iA in the sleep-promoting ON state. When dopamine switches the cells OFF, voltage-dependent currents are attenuated and leak currents augmented. This seesaw form of regulation should be sensitive to perturbations of the neurons' ion channel inventory: depletion of voltage-gated A-type (KV) channels (which predominate in the ON state) should tip the cells towards the OFF state; conversely, loss of leak channels (which predominate in the OFF state) should favour the ON state. To test these predictions, sleep was examined in flies carrying R23E10-GAL4-driven RNAi transgenes for dFB-restricted interference with individual potassium channel transcripts (Pimentel, 2016).

    RNAi-mediated knockdown of two of the five KV channel types of Drosophila (Shaker and Shab) reduced sleep relative to parental controls, while knockdown of the remaining three types had no effect. Biasing the potassium channel repertoire of dFB neurons against A-type conductances thus tilts the neurons' excitable state towards quiescence, causing insomnia, but leaves transient and sustained dopamine responses unaffected. The seemingly counterintuitive conclusion that reducing a potassium current would decrease, not increase, action potential discharge is explained by a requirement for A-type channels in generating repetitive activity of the kind displayed by dFB neurons during sleep. Depleting Shaker from dFB neurons shifted the interspike interval distribution towards longer values, as would be expected if KV channels with slow inactivation kinetics replaced rapidly inactivating Shaker as the principal force opposing the generation of the next spike. These findings identify a potential mechanism for the short-sleeping phenotypes caused by mutations in Shaker, its β subunit Hyperkinetic, or its regulator sleepless (Pimentel, 2016).

    Leak conductances are typically formed by two-pore-domain potassium (K2P) channels. dFB-restricted RNAi of one member of the 11-strong family of Drosophila K2P channels, encoded by the CG8713 gene, increased sleep relative to parental controls; interference with the remaining 10 K2P channels had no effect. Recordings from dFB neurons after knockdown of the CG8713 gene product, which this study termed Sandman, revealed undiminished non-A-type currents in the ON state and intact responses to a single pulse of dopamine but a defective OFF switch: during prolonged dopamine applications, inon-A failed to rise, input resistances and membrane time constants remained at their elevated levels, and the neurons continued to fire action potentials (7 out of 7 cells). Blocking vesicle exocytosis in the recorded cell with botulinum neurotoxin C (BoNT/C) similarly disabled the OFF switch. This, combined with the absence of detectable Sandman currents in the ON state, suggests that Sandman is internalized in electrically active cells and recycled to the plasma membrane when dopamine switches the neurons OFF (Pimentel, 2016).

    Because dFB neurons lacking Sandman spike persistently even after prolonged dopamine exposure, voltage-gated sodium channels remain functional in the OFF state. The difficulty of driving control cells to action potential threshold in this state must therefore be due to a lengthening of electrotonic distance between sites of current injection and spike generation. This lengthening is an expected consequence of a current leak, which may uncouple the axonal spike generator from somatodendritic synaptic inputs or pacemaker currents when sleep need is low (Pimentel, 2016).

    The two kinetically and mechanistically distinct actions of dopamine on dFB neurons-instant, but transient, hyperpolarization and a delayed, but lasting, switch in excitable state-ensure that transitions to vigilance can be both immediate and sustained, providing speedy alarm responses and stable homeostatic control. The key to stability lies in the switching behaviour of dFB neurons, which is driven by dopaminergic input accumulated over time. Unlike bistable neurons, in which two activity regimes coexist for the same set of conductances, dFB neurons switch regimes only when their membrane current densities change. This analysis of how dopamine effects such a change, from activity to silence, has uncovered elements familiar from other modulated systems: simultaneous, antagonistic regulation of multiple conductances; reduction of iA; and modulation of leak currents. Currently little is known about the reverse transition, from silence to activity, except that mutating the Rho-GTPase-activating protein Crossveinless-c locks dFB neurons in the OFF state, resulting in severe insomnia and an inability to correct sleep deficits. Discovering the signals and processes that switch sleep-promoting neurons back ON will hold important clues to the vital function of sleep (Pimentel, 2016).

    Serotonin modulates a depression-like state in Drosophila responsive to lithium treatment

    Major depressive disorder (MDD) affects millions of patients; however, the pathophysiology is poorly understood. Rodent models have been developed using chronic mild stress or unavoidable punishment (learned helplessness) to induce features of depression, like general inactivity and anhedonia. This study reports a three-day vibration-stress protocol for Drosophila that reduces voluntary behavioural activity. As in many MDD patients, lithium-chloride treatment can suppress this depression-like state in flies. The behavioural changes correlate with reduced serotonin (5-HT) release at the mushroom body (MB) and can be relieved by feeding the antidepressant 5-hydroxy-L-tryptophan or sucrose, which results in elevated 5-HT levels in the brain. This relief is mediated by 5-HT-1A receptors in the α-/β-lobes of the MB, whereas 5-HT-1B receptors in the γ-lobes control behavioural inactivity. The central role of serotonin in modulating stress responses in flies and mammals indicates evolutionary conserved pathways that can provide targets for treatment and strategies to induce resilience (Ries, 2017).

    Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila

    Kohlschutter-Tonz syndrome (KTS) is a rare genetic disorder with neurological dysfunctions including seizure and intellectual impairment. Mutations at the Rogdi locus have been linked to development of KTS, yet the underlying mechanisms remain elusive. This study demonstrates that a Drosophila homolog of Rogdi acts as a novel sleep-promoting factor by supporting a specific subset of gamma-aminobutyric acid (GABA) transmission. Rogdi mutant flies displayed insomnia-like behaviors accompanied by sleep fragmentation and delay in sleep initiation. The sleep suppression phenotypes were rescued by sustaining GABAergic transmission primarily via metabotropic GABA receptors or by blocking wake-promoting dopaminergic pathways. Transgenic rescue further mapped GABAergic neurons as a cell-autonomous locus important for Rogdi-dependent sleep, implying metabotropic GABA transmission upstream of the dopaminergic inhibition of sleep. Consistently, an agonist specific to metabotropic but not ionotropic GABA receptors titrated the wake-promoting effects of dopaminergic neuron excitation. Taken together, these data provide the first genetic evidence that implicates Rogdi in sleep regulation via GABAergic control of dopaminergic signaling. Given the strong relevance of GABA to epilepsy, it is proposed that similar mechanisms might underlie the neural pathogenesis of Rogdi-associated KTS (Kim, 2017).

    Neurological disorders caused by single-gene mutations are important genetic models to understand how individual genes execute their roles to support the development and function of the brain, as in the case of Kohlschutter-Tönz syndrome (KTS). KTS patients display developmental delays and psychomotor regression. The most prominent symptoms include amelogenesis imperfecta, early-onset seizures, and intellectual disabilities. Linkage analyses followed by genomic sequencing have revealed that most, if not all, KTS patients have homozygous nonsense, frameshift deletion, or splicing site mutations at the Rogdi locus. ROGDI protein expression is not detectable in affected individuals, indicating that the loss of Rogdi function is responsible for the pathogenesis of KTS. In wild-type human tissues, Rogdi transcripts are ubiquitously expressed while the highest enrichment is evident in adult brain and spinal cord. This observation is consistent with the neurological phenotypes observed in KTS patients (Kim, 2017).

    Given that Rogdi homologs are relatively well conserved in higher eukaryotes, animal models may facilitate understanding of Rogdi-dependent neural processes. In fact, Rogdi was initially identified in a Drosophila genetic screen as a memory-relevant gene and was thus named after one of Pavlov’s dogs. Sequence analyses of Rogdi homologs revealed a putative leucine zipper (ZIP) motif, which could mediate the dimerization of DNA-binding basic ZIP (bZIP) transcription factors. Interestingly, ROGDI proteins localize to the nuclear envelope in cultured human cells, although they lack basic amino acid residues that are typically located at the N-terminus of the ZIP domain and are required for the DNA-binding and nuclear localization of the bZIP transcription factors. Nonetheless, few or no studies have demonstrated the biological activity of Rogdi and genetic models for Rogdi homologs have not been reported yet. Therefore, how Rogdi exerts its physiological roles particularly in the central nervous system and how its mutation leads to the development of KTS are largely unknown (Kim, 2017).

    In the course of genetic studies to elucidate genes and regulatory pathways involved in sleep behaviors, novel sleep mutant alleles were identified in the Drosophila Rogdi gene. This study employed the sleep-promoting effects of Rogdi as a readout of its neural function and demonstrated that Rogdi acts cell-autonomously in GABAergic neurons to enhance metabotropic GABA transmission and thereby sustain sleep. In addition, dopaminergic rescue of Rogdi mutant sleep revealed a novel sleep-regulatory mechanism that functionally links a specific subset of sleep-promoting GABAergic neurons to a wake-promoting dopaminergic pathway. Since epilepsy, a well penetrated phenotype in KTS patients, implicates GABAergic transmission, the current findings provide an important genetic clue to understanding the molecular and neural pathogenesis of KTS (Kim, 2017).

    Modeling of neurological diseases and disease-relevant genes has greatly advanced understanding of the fundamental principles that underlie disease pathogenesis as well as brain function. This study has established the first genetic model of the KTS-associated disease gene Rogdi to demonstrate that Rogdi functions as a novel sleep-promoting factor in GABAergic neurons by promoting GABA transmission. While GABA-dependent sleep regulation via ionotropic GABA receptors have been well documented in Drosophila, the data suggest that GABAergic transmission via metabotropic GABA receptors might be primarily compromised by Rogdi mutation. Furthermore, the wake-promoting DA pathway was identified as a neural locus downstream of Rogdi-dependent GABA signaling given that Rogdi mutant sleep could be rescued by pharmacological or genetic manipulation of dopaminergic transmission. This sleep-regulatory pathway was further supported by the observation that wake-promoting effects of TH-expressing dopaminergic neurons could be selectively titrated by an agonist of metabotropic GABA receptors. Rogdi thus defines a novel pathway coupling these two neurotransmitters to promote baseline sleep in Drosophila as exemplified in other behavioral paradigms across species. On the other hand, Rogdi-dependent GABA transmission might have inhibitory effects on a sleep-promoting neural pathway for sleep homeostasis to suppress recovery sleep after sleep loss (Kim, 2017).

    What is the molecular basis by which Rogdi supports GABAergic transmission and promotes sleep? A possible role of ROGDI as a transcription factor has been suggested by the nuclear localization of human ROGDI protein, particularly in the nuclear envelope of blood mononuclear cells and dermal fibroblasts, and by the conservation of a putatively dimerizing leucine zipper (ZIP) motif among ROGDI homologs. Several lines of evidence, however, argue against this possibility. bZIP transcription factors possess basic residues followed by their ZIP domains whereas ROGDI protein lacks the canonical motif (i.e., basic residues) for DNA-binding activity and nuclear localization. Drosophila ROGDI actually displays its subcellular distribution in both nucleus and cytoplasm of cultured cells or adult fly neurons although the exclusive nuclear localization might not be a prerequisite for transcriptional activities. The crystal structure of human ROGDI protein showed that, unlike other bZIP transcription factors, human ROGDI protein exists as a monomer containing two structurally distinguishable domains (designated as α and β domains, respectively). The α domain exhibits an α-helical bundle that consists of H1, H2, H3, and H6 helices. In fact, the ZIP-like motif in the α domain appears to mediate their intramolecular interactions, contributing to the overall structure and stability of a monomeric ROGDI protein. Based on sequence homology between Drosophila and human ROGDI proteins, it is predicted that Rogdi[del] allele removes the majority of the first helix including the repeated leucine residues in the α domain and the first three strands in the β domain, explaining the instability of ROGDIdel proteins. A smaller but comparable deletion of the ZIP-like motif has been reported in a KTS patient with a splicing mutation in human Rogdi gene. In addition, a functional study in cervical cancer cell lines demonstrated Rogdi effects on cell cycle progression and radio-sensitivity. However, further investigations will be required to understand how these cellular phenotypes could be linked to the molecular and neural function of ROGDI protein (Kim, 2017).

    What will be the relevance of these findings to KTS pathogenesis? Genetic heterogeneity has been reported among KTS patients, indicating that Rogdi-independent genetic mutations could contribute to KTS pathogenesis. A recent study indeed showed that familial mutations in a sodium-citrate transporter gene SLC13A5 are the second genetic cause of KTS. The pathogenic phenotypes commonly found in Rogdi- and SLC13A5-associated KTS gives rise to the intriguing possibility that these two genes might work together to control the intracellular levels of citrate. This idea is further supported by the relevance of citrate metabolism to neurological phenotypes in KTS patients. Neurons are energetically dependent on astrocytes because neurons lack pyruvate carboxylase, an enzyme that converts pyruvate to oxaloacetate in the citric acid cycle. SLC13A5 plays an important role in the transport of glial citrate into neurons to supplement the neuronal citric acid cycle and thereby supply cellular energy. Furthermore, citrate, an intermediate in the citric acid cycle, acts as a precursor of α-ketoglutarate, which can be metabolized to glutamate and GABA, implicating SLC13A5 in the biogenesis of GABA. Consistently, anti-epileptic drugs that elevate GABAergic transmission rescued the seizure phenotypes in SLC13A-associated KTS patients. In addition, this study showed that the pharmacological enhancement of GABAergic transmission by oral administration of GABA-T or GAT inhibitors was sufficient to rescue the short sleep phenotypes in Rogdi mutant flies (Kim, 2017).

    These genetic studies strongly implicate Rogdi function in GABAergic transmission, providing the first clue to understanding the neurological phenotypes observed in KTS patients. Molecular and neural deficits selectively caused by Rogdi mutation might explain why seizures in Rogdi-associated KTS are often resistant to anti-epileptic drugs. Future studies should thus address if Rogdi mutant flies display seizure-like behaviors similarly as in KTS patients and if Rogdi-dependent neural relay of GABAergic transmission controls seizure susceptibility in parallel with baseline sleep. In addition, it will be important to determine whether sleep deficiencies are also observed in KTS patients and whether reduced GABAergic transmission in Rogdi- and, possibly, SLC13A5-associated KTS patients is responsible for their neural dysfunctions, including early-onset seizures. Taken together, this genetic model would constitute an important platform for elucidating the molecular and neural pathogenesis underlying KTS and hint towards a precise development of a therapeutic strategy for KTS in the future (Kim, 2017).

    A higher brain circuit for immediate integration of conflicting sensory information in Drosophila
    Animals continuously evaluate sensory information to decide on their next action. Different sensory cues, however, often demand opposing behavioral responses. How does the brain process conflicting sensory information during decision making? This study shows that flies use neural substrates attributed to odor learning and memory, including the mushroom body (MB), for immediate sensory integration and modulation of innate behavior. Drosophila melanogaster must integrate contradictory sensory information during feeding on fermenting fruit that releases both food odor and the innately aversive odor CO2. Using this framework, this study examined the neural basis for this integration. A local circuit consisting of specific glutamatergic output and PAM dopaminergic input neurons with overlapping innervation in the MB-β'2 lobe region was identified, which integrates food odor and suppresses innate avoidance. Activation of food odor-responsive dopaminergic neurons reduces innate avoidance mediated by CO2-responsive MB output neurons. The study hypothesizes that the MB, in addition to its long recognized role in learning and memory, serves as the insect's brain center for immediate sensory integration during instantaneous decision making (Lewis, 2015).

    Serotonergic modulation enables pathway-specific plasticity in a developing sensory circuit in Drosophila

    How experiences during development cause long-lasting changes in sensory circuits and affect behavior in mature animals are poorly understood. This study establish a novel system for mechanistic analysis of the plasticity of developing neural circuits by showing that sensory experience during development alters nociceptive behavior and circuit physiology in Drosophila larvae. Despite the convergence of nociceptive and mechanosensory inputs on common second-order neurons (SONs), developmental noxious input modifies transmission from nociceptors to their SONs, but not from mechanosensors to the same SONs, which suggests striking sensory pathway specificity. These SONs activate serotonergic neurons to inhibit nociceptor-to-SON transmission; stimulation of nociceptors during development sensitizes nociceptor presynapses to this feedback inhibition. The results demonstrate that, unlike associative learning, which involves inputs from two sensory pathways, sensory pathway-specific plasticity in the Drosophila nociceptive circuit is in part established through feedback modulation. This study elucidates a novel mechanism that enables pathway-specific plasticity in sensory systems (Kaneko, 2017).

    A subset of serotonergic neurons evokes hunger in adult Drosophila

    Hunger is a complex motivational state that drives multiple behaviors. The sensation of hunger is caused by an imbalance between energy intake and expenditure. Although progress is being made, how hunger is represented in the brain and how it coordinates these behavioral responses is not fully understood in any system. This study uses Drosophila melanogaster to identify neurons encoding hunger. A small group of neurons was found that, when activated, induces a fed fly to eat as though it is starved, suggesting that these neurons are downstream of the metabolic regulation of hunger. Artificially activating these neurons also promotes appetitive memory performance in sated flies, indicating that these neurons are not simply feeding command neurons but likely play a more general role in encoding hunger. The neurons relevant for the feeding effect are serotonergic and project broadly within the brain, suggesting a possible mechanism for how various responses to hunger are coordinated. These findings extend the understanding of the neural circuitry that drives feeding and enable future exploration of how state influences neural activity within this circuit (Albin, 2015).

    Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger

    Free-living animals must not only regulate the amount of food they consume but also choose which types of food to ingest. The shifting of food preference driven by nutrient-specific hunger can be essential for survival, yet little is known about the underlying mechanisms. This study identified a dopamine circuit that encodes protein-specific hunger in Drosophila. The activity of these neurons increased after substantial protein deprivation. Activation of this circuit simultaneously promoted protein intake and restricted sugar consumption, via signaling to distinct downstream neurons. Protein starvation triggered branch-specific plastic changes in these dopaminergic neurons, thus enabling sustained protein consumption. These studies reveal a crucial circuit mechanism by which animals adjust their dietary strategy to maintain protein homeostasis (Liu, 2017).

    Beyond satisfying caloric needs, animals must also ingest nutrients that cannot be biosynthesized. The 'specific appetite' hypothesis posits that nutrient-specific hunger drives homeostatic consumption of substances such as protein and salt. Protein is an indispensable macronutrient and is particularly required in anabolic states, such as infancy and pregnancy. Recent studies in Drosophila have identified signaling mechanisms regulating protein hunger, particularly in the context of postmating effects. However, specific circuits encoding protein hunger remain unknown (Liu, 2017).

    Yeast is an ethologically relevant protein food source for Drosophila, containing a negligible amount of sugars. Studies in fruit flies demonstrated that mated females have greater drive for protein and amino acid intake. This study used this potent physiological drive for protein in mated females as an entry point to investigate the neural basis of protein hunger. Yeast-feeding in mated females were assayed after conditional silencing of different neuromodulatory cell groups. Dopamine (DA) neurons were specifically required for yeast consumption and preference. To identify the specific DA neurons mediating this effect, two restricted Gal4 drivers (TH-C-Gal4 and TH-D-Gal4) containing regulatory sequences of the tyrosine hydroxylase gene (TH) were used, and it was found that conditional silencing of the neurons labeled with either driver led to significantly reduced protein preference. These drivers label largely nonoverlapping DA cells, aside from two cells in each protocerebral posterior medial 2 (PPM2) subgroup (Liu, 2017).

    To isolate these PPM2 neurons, an intersectional approach (TH-C-FLP with TH-D-Gal4, was used where FLP encodes flippase) to drive expression of FRT-stop-FRT-mCD8-GFP (a membrane tethered green fluorescent protein). Two PPM2 neurons in each hemisphere were identifed that project ventrally to the 'wedge' neuropil. On the basis of this projection pattern, these two PPM2 neurons were named DA-WED cells. Next, using the FLP-induced intersectional GAL80/GAL4 repression (FINGR) system [TH-D-Gal4, TH-C-FLP, tub-FRT-Gal80-FRT (WED1-Gal4)] to express dendritic versus terminal markers, it was found that the wedge area contains the dendritic field, whereas the two dorsally bifurcating branches contain presynaptic terminals. Intersection between TH-C-Gal4 and another restricted driver TH-F3-Gal4 (15) (WED2-Gal4) also revealed the two DA-WED neurons. These cells were specifically inactivated with the inward-rectifying potassium channel Kir2.1, and substantial inhibition was found of yeast preference and consumption. General hunger, thirst, and salt appetite were not affected when DA-WED neurons were silenced (Liu, 2017).

    It was next asked whether the DA-WED neurons play a general role in homeostatic regulation of protein intake, beyond mated females. Although male flies did not prefer yeast at baseline, they exhibited significant protein preference and consumption after yeast deprivation for 8 days, which was fully suppressed if an amino acid mix was provided during the deprivation period. Inactivating the DA-WED neurons significantly reduced yeast preference and consumption in protein-deprived male flies. Conversely, conditional activation of these cells with the heat-activated cation channel dTrpA1 induced yeast preference and consumption in males. Similar data were obtained for virgin females. Conditional silencing or activation of DA-WED neurons reduced or increased protein consumption, respectively, over a range of internal protein-hunger states in both mated female and male flies, it was decided to focus on male flies for subsequent experiments to avoid postmating effects. Reducing DA levels in DA-WED neurons by knocking down the neuronal specific isoform of TH in these cells decreased yeast intake in protein-starved male flies (Liu, 2017).

    To investigate whether the activity of the DA-WED neurons correlates with protein need, perforated patch-clamp recordings were performed from these neurons. After yeast deprivation, the spontaneous action potential (AP) firing rate of DA-WED neurons increased about fourfold compared to controls. Moreover, evoked AP firing rates were higher at all measured depolarizing currents after yeast deprivation. Protein starvation did not significantly alter the resting membrane potential or input resistance of these cells. Cytosolic Ca2+ concentrations in these cells (but not in nearby PPM3 DA neurons) was substantially increased after yeast deprivation when monitored with the CaLexA (calcium-dependent nuclear import of LexA) activity reporter system. This increase in intracellular Ca2+ concentration was suppressed when an amino acid mix was provided in the protein-deficient diet. This effect was specific for protein, as general starvation did not affect CaLexA signal in the DA-WED neurons. Similar data were obtained with GCaMP imaging, as an alternative method to measure cytosolic Ca2+ levels (Liu, 2017).

    What component of protein serves to signal protein satiety to the DA-WED neurons? It was hypothesized that a specific amino acid may play this role. Flies were first subjected to protein deprivation, but provided individual amino acids in the diet, and then monitored the activity of DA-WED neurons using CaLexA. Tryptophan and glutamine (Gln) supplementation suppressed the enhanced activity of DA-WED neurons induced by protein deprivation, and proline and glutamate supplementation showed a trend toward this effect. Next, whether supplementation of these four individual amino acids modulated homeostatic regulation of yeast intake was tested. Yeast intake was significantly inhibited when Gln was added back to a protein-deficient diet. Conversely, an amino acid mix lacking only Gln failed to suppress the increase in DA-WED activity and was less effective at inhibiting protein consumption after yeast deprivation. It was also found that Gln levels in the hemolymph were significantly reduced after protein deprivation. Together, these data suggest that Gln in particular may be important for signaling protein deficiency (Liu, 2017).

    Despite strong preference for sucrose at baseline, yeast deprivation caused flies to decrease their consumption of this nutrient. Because DA-WED neurons are activated after substantial protein deprivation, it was hypothesized that they also play a direct role in reducing sucrose intake under these conditions. Silencing DA-WED neurons with Kir2.1 significantly enhanced sugar intake after protein deprivation, whereas activating these neurons reduced sucrose consumption (Liu, 2017).

    The mechanisms underlying the opposing effects of DA-WED neurons on sucrose and yeast intake was investigated. There are four DA receptors in Drosophila encoded by DopR1, DopR2, D2R, and DopEcR. Both DopR1 and DopR2 mutants exhibited a reduced preference for yeast after protein deprivation in a two-choice assay, which could reflect either a reduced preference for protein or a greater preference for sucrose. Thus, focus was placed on these two receptors as potential targets acting downstream of the DA-WED neurons. In one-choice assays, DopR2 mutants exhibited significantly reduced yeast intake, whereas DopR1 mutants displayed a robust increase in sucrose feeding. Mutations in DopR2 and DopR1 also suppressed the increase in yeast consumption and decrease in sucrose feeding triggered by activating DA-WED neurons, respectively. These findings were confirmed by pan-neuronal knockdown of DopR1 or DopR2 (nsyb-Gal4>UAS-DopR1-miR or nsyb-Gal4>UAS-DopR2-miR), which yielded similar results. It was therefore hypothesized that DopR2 and DopR1 act in discrete neuronal targets of the DA-WED cells to regulate protein and sugar feeding, respectively (Liu, 2017).

    To identify putative candidate circuits acting downstream of the DA-WED cells, a GRASP (GFP reconstitution across synaptic partners)-based screen was performed with Rubin Gal4 drivers derived from DopR2 and DopR1 enhancer sequences to assess connectivity with the DA-WED neurons. Among DopR2-derived driver lines, only the R70G12-Gal4 line exhibited GRASP signal at the DA-WED terminal branches (specifically, the medial branch). Moreover, knockdown of DopR2 with R70G12-Gal4 significantly reduced yeast intake after protein starvation, without affecting general hunger or sucrose consumption in yeast-deprived animals. This reduction in protein appetite in R70G12-Gal4>UAS-DopR2-miR flies was observed over a range of protein-hunger states in both mated female and male flies. To identify the specific cells in this driver that contact the DA-WED neurons, a multicolor flip out (MCFO) experiment was performed. A neuron whose cell body is located in the posterior medial protocerebrum and sends its projections first to the fan-shaped body (FB), before extending more anteriorly to the lateral accessory lobe (LAL) area was identified where the GRASP signal was observed. These neurons were named FB-LAL cells based on this projection pattern. An independent driver R75B10-Gal4 was identified that also labeled the FB-LAL neuron and displayed GRASP signal at the medial branch of the DA-WED neurons. Knockdown of DopR2 with R75B10-Gal4 decreased yeast intake in protein-starved flies. As expected, intersection between R70G12-Gal4 and R75B10-Gal4 drivers (R70G12-Gal4, R75B10-LexA>LexAop-FLP, UAS-FRT-stop-FRT-mCD8-GFP) revealed two pairs of FB-LAL neurons. Silencing these cells reduced yeast intake in protein-starved flies, whereas activating these cells induced elevated yeast consumption in male flies. Activation of DA-WED neurons expressing ATP (adenosine 5'-triphosphate)-gated P2X2 receptors induced a substantial increase in GCaMP signal in the cell bodies of downstream FB-LAL neurons (Liu, 2017).

    By contrast, among the DopR1-derived lines, the R72B03-Gal4 driver demonstrated GRASP signal at the DA-WED lateral branches. Knockdown of DopR1 with the R72B03-Gal4 driver triggered an increase in sucrose intake in protein-deprived flies, without affecting general hunger or yeast intake after protein starvation. MCFO analysis using R72B03-Gal4 revealed a small subset of neurons in the posterior lateral protocerebrum (PLP) that send their projections locally to the ventrolateral and superior neuropils, so these cells were named PLP neurons. PLP neurons were also labeled by another driver line R32A06-Gal4. Similar to R72B03-Gal4, R32A06-Gal4 exhibited GRASP signal at the lateral branch of DA-WED neurons. MCFO analysis with the new R32A06-Gal4 driver revealed cells whose cell body location and projection pattern match that seen with the PLP neuron identified in the R72B03-Gal4 driver. Knockdown of DopR1 with R32A06-Gal4 increased sucrose intake in protein-starved flies. Silencing or activating these PLP neurons with R72B03-Gal4 or R32A06-Gal4 led to a reduction or an increase, respectively, of sucrose intake (Liu, 2017).

    Following substantial deprivation of an essential nutrient, animals must engage in persistent behavior aimed at replenishing it. Plasticity of relevant neural circuits may underlie the persistent drive for motivated behaviors. To address whether the DA-WED neurons undergo protein hunger-dependent plastic changes, the morphology of their terminal branches was assessed using mCD8-GFP and a GFP-tagged version of the synaptic vesicle-associated protein synaptotagmin (Syt-GFP). The medial, but not the lateral, branches were significantly elongated following protein deprivation, which was suppressed when Gln was selectively restored in the protein-deficient diet. Similar data were obtained for the number and total volume of Syt-GFP+ puncta. To address whether the number of active zones also changed in the medial branches of DA-WED neurons after protein starvation, a truncated fragment of the active-zone marker Bruchpilot (Brp-short) was expressed in these cells. The number of BRP+ puncta in the medial, but not lateral, branch was significantly elevated when flies were protein-deprived, and this increase showed a trend toward being inhibited by Gln in the protein-deficient diet. GRASP was used to determine whether protein starvation altered the connectivity between the DA-WED cells and their downstream targets. GRASP signal between the medial branch of the DA-WED neurons and the FB-LAL cells, but not the lateral branch and PLP neurons, was substantially elevated with protein starvation, and this increase was again suppressed if Gln was provided in the protein-deficient diet (Liu, 2017).

    To characterize the functional consequences of the plastic changes in the DA-WED neurons, sharp intracellular current-clamp recordings of the FB-LAL cells were performed to measure frequency and amplitude of postsynaptic potentials (PSPs). Protein deprivation induced a substantial increase in the frequency of PSPs in the FB-LAL neurons. Moreover, examination of the distribution of PSP amplitudes revealed the presence of high-amplitude PSPs solely in yeast-deprived animals. This population of high-amplitude PSPs likely reflects evoked responses seen only with protein deprivation and suggests that downstream signaling is markedly enhanced after protein starvation. Knockdown of DopR2 in FB-LAL cells significantly reduced the number of high-amplitude PSPs in yeast-deprived animals, consistent with a model wherein the DA-WED neurons signal via DopR2 on the FB-LAL cells to promote protein consumption (Liu, 2017).

    Whether the plastic changes in the medial branches of the DA-WED cells led to a change in feeding behavior was examined. dTrpA1 was used to activate the DA-WED cells and yeast or sucrose consumption was assessed at different time points after cessation of heat treatment. dTrpA1 activation of the DA-WED neurons resulted in a prolonged increase in yeast consumption lasting at least 6 hours and induced persistent plastic changes in the medial, but not lateral, branches as revealed by Syt-GFP staining. In contrast, although sucrose intake was reduced immediately after dTrpA1 activation, this response did not persist and was no longer present within 1 hour. After 24 hours, both yeast consumption and terminal morphology of DA-WED neurons were indistinguishable from control flies. Together, these findings demonstrate branch-specific plastic changes of the DA-WED neurons in response to substantial protein deprivation, providing a mechanism for the persistent hunger for protein, but not sugar, under these conditions (Liu, 2017).

    Appropriate regulation of food consumption is essential for the survival of organisms that must navigate environments with variable and uncertain food availability and quality. A number of studies have investigated how animals reject diets devoid of essential amino acids and how monoaminergic and TOR-S6K (target of rapamycin-S6 kinase) signaling in Drosophila regulates mating-induced protein feeding. However, little is known about the circuit mechanisms mediating the homeostatic regulation of protein intake. This study suggests that protein hunger is encoded by the DA-WED neurons, providing a path toward dissecting these mechanisms. The findings suggest that glutamine, directly or indirectly, regulates the activity of these neurons, thus modulating behavioral responses to protein deprivation (Liu, 2017).

    The data suggest that the DA-WED neurons play a crucial role in homeostatic control of protein intake, functioning on multiple time scales to restore protein balance. Shortly after substantial protein deprivation, the DA-WED neurons act to mediate the behavioral switch between consumption of a food source preferred at baseline (sucrose) and the deprived nutrient (protein), by activating a dedicated 'protein' feeding circuit, while simultaneously inhibiting a 'sugar' feeding circuit. Over a longer time frame, the DA-WED cells undergo branch-specific plastic changes that underlie the selective and persistent hunger for protein under these conditions. In the wild, these actions may correspond to promoting greater selectivity for protein in an initial foraging response, followed by maintenance of protein consumption after the protein food source has been identified (Liu, 2017).

    Given that branch-specific plasticity has generally been described in postsynaptic dendrites, the finding that the presynaptic terminals of the DA-WED neurons exhibit this phenomenon is highly unusual. The mechanisms underlying this process are currently unclear but may depend on extrinsic neuromodulatory influences that differentially regulate potential for plasticity of the distinct presynaptic terminals of the DA-WED cells. Further characterization of these and related circuit mechanisms should help delineate the fundamental principles governing protein-specific hunger. A better understanding of how animals choose to consume protein may also have implications for the treatment of obesity (Liu, 2017).

    A single pair of serotonergic neurons counteracts serotonergic inhibition of ethanol attraction in Drosophila

    Attraction to ethanol is common in both flies and humans, but the neuromodulatory mechanisms underlying this innate attraction are not well understood. This study dissected the function of the key regulator of serotonin signaling - the serotonin transporter - in innate olfactory attraction to ethanol in Drosophila melanogaster. A mutated version of the serotonin transporter was generated that prolongs serotonin signaling in the synaptic cleft and is targeted via the Gal4 system to different sets of serotonergic neurons. Four serotonergic neurons were identified that inhibit the olfactory attraction to ethanol, and two additional neurons were identified that counteract this inhibition by strengthening olfactory information. These results reveal that compensation can occur on the circuit level and that serotonin has a bidirectional function in modulating the innate attraction to ethanol. Given the evolutionarily conserved nature of the serotonin transporter and serotonin, the bidirectional serotonergic mechanisms delineate a basic principle for how random behavior is switched into targeted approach behavior (Xu, 2016).

    Dynamics of memory-guided choice behavior in Drosophila

    Memory retrieval requires both accuracy and speed. Olfactory learning of the fruit fly Drosophila melanogaster serves as a powerful model system to identify molecular and neuronal substrates of memory and memory-guided behavior. The behavioral expression of olfactory memory has traditionally been tested as a conditioned odor response in a simple T-maze, which measures the result, but not the speed, of odor choice. This study developed multiplexed T-mazes that allow video recording of the choice behavior. Automatic fly counting in each arm of the maze visualizes choice dynamics. Using this setup, it was shown that the transient blockade of serotonergic neurons slows down the choice, while leaving the eventual choice intact. In contrast, activation of the same neurons impairs the eventual performance leaving the choice speed unchanged. This new apparatus contributes to elucidating how the speed and the accuracy of memory retrieval are implemented in the fly brain (Ichinose, 2016).

    Coordinated and compartmentalized neuromodulation shapes sensory processing in Drosophila

    Learned and adaptive behaviors rely on neural circuits that flexibly couple the same sensory input to alternative output pathways. This study shows that the Drosophila mushroom body functions like a switchboard in which neuromodulation reroutes the same odor signal to different behavioral circuits, depending on the state and experience of the fly. Using functional synaptic imaging and electrophysiology, it was shown that dopaminergic inputs to the mushroom body modulate synaptic transmission with exquisite spatial specificity, allowing individual neurons to differentially convey olfactory signals to each of their postsynaptic targets. Moreover, the dopaminergic neurons function as an interconnected network, encoding information about both an animal's external context and internal state to coordinate synaptic plasticity throughout the mushroom body. These data suggest a general circuit mechanism for behavioral flexibility in which neuromodulatory networks act with synaptic precision to transform a single sensory input into different patterns of output activity (Cohn, 2015).

    Neural circuits for long-term water-reward memory processing in thirsty Drosophila

    The intake of water is important for the survival of all animals and drinking water can be used as a reward in thirsty animals. This study found that thirsty Drosophila melanogaster can associate drinking water with an odour to form a protein-synthesis-dependent water-reward long-term memory (LTM). Furthermore, the reinforcement of LTM requires water-responsive dopaminergic neurons projecting to the restricted region of mushroom body (MB) β' lobe, which are different from the neurons required for the reinforcement of learning and short-term memory (STM). Synaptic output from α'β' neurons is required for consolidation, whereas the output from γ and αβ neurons is required for the retrieval of LTM. Finally, two types of MB efferent neurons retrieve LTM from γ and αβ neurons by releasing glutamate and acetylcholine, respectively. These results therefore cast light on the cellular and molecular mechanisms responsible for processing water-reward LTM in Drosophila (Shyu, 2017).

    Serotonergic network in the subesophageal zone modulates the motor pattern for food intake in Drosophila

    The functional organization of central motor circuits underlying feeding behaviors is not well understood. This study combined electrophysiological and genetic approaches to investigate the regulatory networks upstream of the motor program underlying food intake in the Drosophila larval central nervous system. The serotonergic network of the CNS was found to be capable of setting the motor rhythm frequency of pharyngeal pumping. Pharmacological experiments verified that modulation of the feeding motor pattern is based on the release of serotonin. Classical lesion and laser based cell ablation indicated that the serotonergic neurons in the subesophageal zone represent a redundant network for motor control of larval food intake (Schoofs, 2017).

    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 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).

    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. Fmn (dDAT; Dopamine transporter) mutants displayed hyperactivity in response to mechanical shocks, implying a positive-acting role for DA in controlling environmentally induced arousal. 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. Finally, transient activation of DA neurons in hyperactive flies inhibited locomotion. 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, 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. 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 (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, 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 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).

    Caffeine promotes wakefulness via dopamine signaling in Drosophila
    Caffeine is the most widely-consumed psychoactive drug in the world, but the understanding of how caffeine affects brains is relatively incomplete. Most studies focus on effects of caffeine on adenosine receptors, but there is evidence for other, more complex mechanisms. In the fruit fly Drosophila melanogaster, which shows a robust diurnal pattern of sleep/wake activity, caffeine reduces nighttime sleep behavior independently of the one known adenosine receptor. This study shows that dopamine is required for the wake-promoting effect of caffeine in the fly, and that caffeine likely acts presynaptically to increase dopamine signaling. A cluster of neurons, the paired anterior medial (PAM) cluster of dopaminergic neurons, were identified as the ones relevant for the caffeine response. PAM neurons show increased activity following caffeine administration, and promote wake when activated. Also, inhibition of these neurons abrogates sleep suppression by caffeine. While previous studies have focused on adenosine-receptor mediated mechanisms for caffeine action, this study identifies a role for dopaminergic neurons in the arousal-promoting effect of caffeine (Nall, 2016).

    Identified serotonin-releasing neurons induce behavioral quiescence and suppress mating in Drosophila
    Animals show different levels of activity that are reflected in sensory responsiveness and endogenously generated behaviors. Biogenic amineshave been determined to be causal factors for these states of arousal. It is well established that, in Drosophila, dopamine and octopamine promote increased arousal. However, little is known about factors that regulate arousal negatively and induce states of quiescence. Moreover, it remains unclear whether global, diffuse modulatory systems comprehensively affecting brain activity determine general states of arousal. Alternatively, individual aminergic neurons might selectively modulate the animals' activity in a distinct behavioral context. This study shows that artificially activating large populations of serotonin-releasing neurons induces behavioral quiescence and inhibits feeding and mating. The study systematically narrows down a role of serotonin in inhibiting endogenously generated locomotor activity to neurons located in the posterior medial protocerebrum. Neurons of this cell cluster were identified that suppress mating, but not feeding behavior. These results suggest that serotonin does not uniformly act as global, negative modulator of general arousal. Rather, distinct serotoninergic neurons can act as inhibitory modulators of specific behaviors (Pooryasin, 2015).

    Pre- and postsynaptic role of Dopamine D2 Receptor DD2R in Drosophila olfactory associative learning

    Dopaminergic neurons in Drosophila play critical roles in diverse brain functions such as motor control, arousal, learning, and memory. Using genetic and behavioral approaches, it has been firmly established that proper dopamine signaling is required for olfactory classical conditioning (e.g., aversive and appetitive learning). Dopamine mediates its functions through interaction with its receptors. There are two different types of dopamine receptors in Drosophila: 1) Dopamine 1-like, including Dopamine 1-like receptor 1 and Dopamine 1-like receptor 2 (DDR2, the subject of this report) and 2) Dopamine 2-like receptor. Currently, no study has attempted to characterize the role of DD2R in Drosophila learning and memory. Using a DD2R-RNAi transgenic line, this study has examined the role of DD2R, expressed in dopamine neurons (i.e., the presynaptic DD2R autoreceptor), in larval olfactory learning. The function of postsynaptic DD2R expressed in mushroom body (MB) was also studied as MB is the center for Drosophila learning, with a function analogous to that of the mammalian hippocampus. These results showed that suppression of presynaptic DD2R autoreceptors impairs both appetitive and aversive learning. Similarly, postsynaptic DD2R in MB neurons appears to be involved in both appetitive and aversive learning. The data confirm, for the first time, that DD2R plays an important role in Drosophila olfactory learning (Qi, 2014).

    Dopamine (DA) is an important neurotransmitter mediating a variety of brain functions including locomotion, reward, awareness, learning and memory, and cognition. Genetic and pharmacological studies revealed that the dopaminergic system in the fruit fly Drosophila melanogaster plays multiple roles in motor function and associative learning. Using the sophisticated genetic tools available for the fruit fly, it has been firmly established that release of dopamine is required for associative learning in Drosophila adults and larvae. Dopaminergic neural circuits mediating olfactory learning have been also characterized in the fruit fly brain (Qi, 2014).

    DA mediates its physiological functions through interaction with its receptors. Analysis of the primary structure of the DA receptors revealed that those receptors belong to the G-protein coupled receptor (GPCR) family. Generally, DA receptors can be divided into two families in vertebrates. The D1-like receptor family stimulates cAMP production by activation of the receptor-coupled Gs subunit of G proteins. The D2-like receptor family belongs to the pertussis toxin (PTX)-sensitive G protein (i.e., Gi and Go)-coupled receptor (GPCR) superfamily. Therefore, actions of D2-like receptor members have been characterized as inhibitory. In relation to regulation of DA signaling, DA neurons were found to possess receptors for their own transmitter, dopamine, at their synaptic nerve terminals. These DA autoreceptors (autoR) function as self-inhibitory regulators (Qi, 2014).

    In Drosophila, there are four DA receptors (dDA1, DAMB, DopEcR, and DD2R) that have been cloned and characterized. Two DA receptors (dDA1, DAMB) were cloned first and appear to be members of the D1-like receptor family on the basis of their ability to stimulate adenylyl cyclase (AC) in a heterologous expression system. In contrast, only one Drosophila DA receptor DD2R gene has been identified. Functional expression of the DD2R gene in HEK293 cells indicated that DA caused a marked decrease in forskolin-induced cAMP level, indicating that DD2R belongs to the inhibitory D2-like receptor family. Interestingly, two recent studies confirmed the existence of DA autoreceptors in Drosophila (Qi, 2014).

    Olfactory associative learning in adult flies requires expression of Drosophila D1 receptor dDA1 in the mushroom body, the anatomical center for learning and memory. The dDA1 mutant dumb showed impaired appetitive learning as well as aversive learning. These impaired learning behaviors were fully rescued by expression of the wild-type dDA1 transgene in MB neurons in mutant flies, further confirming the role of Drosophila D1-like receptors in learning. However, no previous study has attempted to characterize the role of D2-like DD2R in Drosophila learning and memory. Interestingly, there was one study showing that a D2 agonist eticlopride did not disrupt visual learning (e.g., T maze assay) in adult flies (Qi, 2014).

    Drosophila larvae carrying DD2R-RNAi transgene were used to examine the role of D2-like receptors in associative learning. Two different types of tissue-specific drivers were used to examine both presynaptic D2 autoreceptors and postsynaptic D2 receptors. Dopaminergic-specific driver TH-Gal4 was used to induce DD2R-RNAi expression in DA neurons. Since the target of dopaminergic innervation is the mushroom body (MB), the center for learning and memory in Drosophila, MB-specific drivers (201Y-Gal4, 30Y-Gal4) were used to down-regulate postsynaptic DD2R combined with DD2R-RNAi transgene. The results showed that both presynaptic DD2R autoreceptors and postsynaptic receptors are required for aversive and appetitive olfactory learning in Drosophila larvae (Qi, 2014).

    The Drosophila D2 receptor DD2R has been shown to play an important role in locomotion, aggression, and neuroprotection. Interestingly, no study has shown whether Drosophila DD2R is involved in learning and memory, although dopaminergic (DA) neural circuits and D1 receptors are known to mediate Drosophila aversive learning. The present study, for the first time, demonstrated that DD2R is involved in olfactory associative learning in Drosophila larvae. Further, we showed that both presynaptic and postsynaptic DD2Rs mediate aversive and appetitive learning in the fly larvae as down-regulation of DD2R in DA and mushroom body (MB) neurons resulted in impaired olfactory learning (Qi, 2014).

    Multiple studies have proved that dopamine signaling is necessary in Drosophila aversive learning. However, it is uncertain whether dopamine signaling is involved in appetitive learning. Several laboratories reported that DA signaling is not necessary for appetitive learning, which is mediated by another biogenic amine, octopamine In contrast, DA signaling has been shown to be necessary for appetitive learning; inhibition of DA release resulted in reduced appetitive learning. Furthermore, D1 receptor mutants (e.g., dDA1) showed impaired appetitive learning (Qi, 2014).

    This study has demonstrated that dopamine mediates not only aversive learning, but also appetitive learning. Both learning behaviors are impaired when DD2R-RNAi is expressed in DA neurons or in MB neurons. Interestingly, aversive learning was completely impaired, while appetitive learning was only partially impaired when DD2R-RNAi was expressed in DA neurons. A possible explanation is that the effect of DD2R-RNAi is partial as RNAi down-regulates the target gene expression. Another possibility is that DA is not the only modulatory neurotransmitter mediating appetitive learning; another biogenic amine, octopamine, is involved in appetitive learning. Therefore, octopamine can mediate appetitive learning to a certain extent even if DA signaling is impaired. In contrast, no modulatory neurotransmitter other than DA is known to be involved in aversive learning (Qi, 2014).

    Reduced locomotion due to expression of DD2R-RNAi has been reported. The current findings do not support that result since the larvae carrying DD2R-RNAi showed no changes in sensory and motor function, compared to WT and control fly strains. This discrepancy can be explained through the following reasons. first, it may be related to developmental-specific effects. This study used third-instar larvae while adult flies were used by Draper. Second, there are differences in locomotion assays. Draper quantified total activity counts, amount of time active, and number of activity-rest bouts. In the current study, crawling speed was measured. Third, DD2R-RNAi expression patterns are different. Draper used Act5C-Gal or elav-Gal4 to express DD2R-RNAi ubiquitously or pan-neuronally, respectively. In contrast, DD2R-RNAi was only expressed in DA or MB neurons in the current study. Therefore, neural circuits affected by DD2R-RNAi can be different, resulting in different behaviors. It is also possible that expression level of DD2R-RNAi is different due to different drivers (Qi, 2014).

    Since the identification of Drosophila D2 receptor DD2R, two studies have revealed the autoreceptor function of DD2R. D2R agonists have been reported to reduce DA release in the Drosophila larval central nervous system. It was also shown that DD2R autoreceptors suppress excitability of DA neurons in Drosophila primary neuronal cultures. This study showed that DD2R is involved in mediating both appetitive and aversive olfactory learning. DD2R-RNAi in DA neurons down-regulates DD2R autoreceptor function. Thus excitability of DA neurons is increased, leading to an increase of DA release. Our results indicate that excessive DA release impairs olfactory learning. Indeed, it has been shown that olfactory learning is impaired in Drosophila DA transporter mutant fumin, likely due to increased synaptic DA levels. In contrast, a lack of DA release is known to cause impaired learning in Drosophila larvae. Therefore, it appears that homeostatic regulation of DA release by DD2R is important for both appetitive and aversive olfactory learning as either too much or too little synaptic DA causes impaired learning. Taking these facts into consideration, a model is proposed to explain the role of presynaptic DD2R autoreceptors. Presynaptic DD2R autoreceptors suppress release of DA at the presynaptic terminals in the MB. If presynaptic DD2R function is suppressed, then more DA is released into MB neurons. Increased DA tone in the MB impairs both aversive and appetitive learning behaviors (Qi, 2014).

    This study also showed that olfactory learning in Drosophila larvae is impaired by down-regulation of postsynaptic DD2R in MB neurons. As the role of DD2R is inhibitory, the effects of DD2R-RNAi in MB neurons are expected to increase neuronal excitability, and thus olfactory learning is impaired by hyperexcitability in MB neurons. This observation might not be consistent with the physiological findings that learning and memory are mediated by enhanced neuronal excitability and synaptic transmission. Such well-known examples are long-term facilitation (LTF) and long-term potentiation (LTP). In this study, DD2R-RNAi is expressed throughout the larval stage. Therefore, hyperexcitability is chronic and thus this increased baseline activity interferes with coding new information in the MB. Indeed, olfactory learning is impaired in Drosophila by the chronic increase of excitatory cholinergic synaptic transmission due to the phosphodiesterase gene dunce mutation, resulting in increased cAMP levels. Taken together, temporal increases in excitability are key physiological changes underlying associative learning and thus DD2R-RNAi interferes with this change by inducing chronic hyperexcitability in MB neurons (Qi, 2014).

    In addition to DD2R, there are Drosophila D1-like receptors (dDA1 and DAMB) that are known to be highly expressed in MB neurons. In fact, dDA1 null mutants showed defects in olfactory learning. Since D1-like receptors increase neuronal excitability via the cAMP-PKA signaling pathway, dDA1 mutant MB neurons are less depolarized when DA is released at the synaptic terminal in the MB, and thus cannot mediate olfactory learning. Proper excitability of MB neurons should be maintained by balancing actions of D1- and D2-like receptors in MB neurons (Qi, 2014).

    It has been proposed that the adenylyl cyclase gene rutabaga in MB is a coincidence detector for CS and US in Drosophila olfactory learning and memory. Therefore, on the basis of the current results, a model is proposed to explain postsynaptic mechanisms underlying aversive and appetitive learning. Postsynaptic DD2Rs in MBNs inhibit neuronal excitability while dDA1 stimulates neural circuits associating CS with US in MB. DA receptors dDA1 and DD2R regulate AC in MB neurons in the opposite direction to maintain homeostatic balance of MB neuronal excitability, which is an important physiological element for Drosophila larval olfactory learning (Qi, 2014).

    In conclusion, this study examined the role of D2-like receptor DD2R in Drosophila olfactory associative learning. The results showed that suppression of presynaptic DD2R autoreceptors impairs both appetitive and aversive learning. Similarly, postsynaptic DD2R in MB neurons appears to be involved in both appetitive and aversive learning (Qi, 2014).

    The data strongly support the hypothesis that presynaptic DD2R autoreceptors suppress release of DA at the presynaptic terminals in the MB. If presynaptic DD2R function is suppressed, then more DA is released. Increasing DA tone to MB neurons impairs both aversive and appetitive learning behaviors. Postsynaptically, DD2R-RNAi impaired olfactory associative learning most likely by inducing chronic hyperexcitability in MB neurons. Therefore, the role of postsynaptic DD2R is to maintain the proper excitability in MB neurons during learning. Taken together, this study, for the first time, demonstrated that DD2R plays an important role in Drosophila olfactory associative learning (Qi, 2014).

    Suppression of dopamine neurons mediates reward

    Massive activation of dopamine neurons is critical for natural reward and drug abuse. In contrast, the significance of their spontaneous activity remains elusive. In Drosophila melanogaster, depolarization of the protocerebral anterior medial (PAM) cluster dopamine neurons en masse signals reward to the mushroom body (MB) and drives appetitive memory. Focusing on the functional heterogeneity of PAM cluster neurons, this study identified that a single class of PAM neurons, PAM-γ3, mediates sugar reward by suppressing their own activity. PAM-γ3 is selectively required for appetitive olfactory learning, while activation of these neurons in turn induces aversive memory. Ongoing activity of PAM-γ3 gets suppressed upon sugar ingestion. Strikingly, transient inactivation of basal PAM-γ3 activity can substitute for reward and induces appetitive memory. Furthermore, the satiety-signaling neuropeptide Allatostatin A (AstA) is a key mediator that conveys inhibitory input onto PAM-γ3. These results suggest the significance of basal dopamine release in reward signaling and reveal a circuit mechanism for negative regulation (Yamagata, 2016)

    References

    Albin, S.D., Kaun, K.R., Knapp, J.M., Chung, P., Heberlein, U. and Simpson, J.H. (2015). A subset of serotonergic neurons evokes hunger in adult Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 26344091

    Aso, Y. and Rubin, G.M. (2016). Dopaminergic neurons write and update memories with cell-type-specific rules. Elife [Epub ahead of print]. PubMed ID: 27441388

    Bednářová, A., Hanna, M.E., Rakshit, K., O'Donnell, J.M. and Krishnan, N. (2017). Disruption of dopamine homeostasis has sexually dimorphic effects on senescence characteristics of Drosophila melanogaster. Eur J Neurosci [Epub ahead of print]. PubMed ID: 28112452

    Berry, J. A., Cervantes-Sandoval, I., Nicholas, E. P. and Davis, R. L. (2012). Dopamine is required for learning and forgetting in Drosophila. Neuron 74: 530-542. PubMed ID: 22578504

    Berry, J.A., Cervantes-Sandoval, I., Chakraborty, M. and Davis, R.L. (2015). Sleep facilitates memory by blocking dopamine neuron-mediated forgetting. Cell 161(7):1656-67. PubMed ID: 26073942

    Blenau, W., Daniel, S., Balfanz, S., Thamm, M. and Baumann, A. (2017). Dm5-HT2B: Pharmacological characterization of the fifth serotonin receptor subtype of Drosophila melanogaster. Front Syst Neurosci 11: 28. PubMed ID: 28553207

    Borycz, J., Borycz, J. A., Loubani, M. and Meinertzhagen, I. A. (2002). tan and ebony genes regulate a novel pathway for transmitter metabolism at fly photoreceptor terminals. J. Neurosci. 22(24): 10549-57. Medline abstract: 12486147

    Boto, T., Louis, T., Jindachomthong, K., Jalink, K. and Tomchik, S. M. (2014). Dopaminergic modulation of cAMP drives nonlinear plasticity across the Drosophila mushroom body lobes. Curr Biol 24: 822-831. PubMed ID: 24684937

    Chen, S. L., Chen, Y. H., Wang, C. C., Yu, Y. W., Tsai, Y. C., Hsu, H. W., Wu, C. L., Wang, P. Y., Chen, L. C., Lan, T. H. and Fu, T. F. (2017). Active and passive sexual roles that arise in Drosophila male-male courtship are modulated by dopamine levels in PPL2ab neurons. Sci Rep 7: 44595. PubMed ID: 28294190

    Cohn, R., Morantte, I. and Ruta, V. (2015). Coordinated and compartmentalized neuromodulation shapes sensory processing in Drosophila. Cell 163: 1742-1755. PubMed ID: 26687359

    Donlea, J. M., Thimgan, M. S., Suzuki, Y., Gottschalk, L. and Shaw, P. J. (2011). Inducing sleep by remote control facilitates memory consolidation in Drosophila. Science 332: 1571-1576. PubMed ID: 21700877

    Donlea, J. M., Pimentel, D. and Miesenbock, G. (2014). Neuronal machinery of sleep homeostasis in Drosophila. Neuron 81: 860-872. PubMed ID: 24559676

    Dylla, K. V., Raiser, G., Galizia, C. G. and Szyszka, P. (2017). Trace conditioning in Drosophila induces associative plasticity in mushroom body kenyon cells and dopaminergic neurons. Front Neural Circuits 11: 42. PubMed ID: 28676744

    Gorostiza, E. A., Colomb, J. and Brembs, B. (2016). A decision underlies phototaxis in an insect.Open Biol 6(12): 160229. PubMed ID: 28003472

    Freyberg, Z., et al. (2016). Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain. Nat Commun 7: 10652. PubMed ID: 26879809

    Hidalgo, S., Molina-Mateo, D., Escobedo, P., Zarate, R. V., Fritz, E., Fierro, A., Perez, E. G., Iturriaga-Vasquez, P., Reyes-Parada, M., Varas, R., Fuenzalida-Uribe, N. and Campusano, J. M. (2017). Characterization of a novel Drosophila SERT mutant: insights on the contribution of the serotonin neural system to behaviors. ACS Chem Neurosci [Epub ahead of print]. PubMed ID: 28665105

    Ichinose, T. and Tanimoto, H. (2016). Dynamics of memory-guided choice behavior in Drosophila. Proc Jpn Acad Ser B Phys Biol Sci 92: 346-357. PubMed ID: 27725473

    Kaneko, T., Macara, A. M., Li, R., Hu, Y., Iwasaki, K., Dunnings, Z., Firestone, E., Horvatic, S., Guntur, A., Shafer, O. T., Yang, C. H., Zhou, J. and Ye, B. (2017). Serotonergic modulation enables pathway-specific plasticity in a developing sensory circuit in Drosophila. Neuron 95(3): 623-638.e624. PubMed ID: 28712652

    Kim, M., Jang, D., Yoo, E., Oh, Y., Sonn, J. Y., Lee, J., Ki, Y., Son, H. J., Hwang, O., Lee, C., Lim, C. and Choe, J. (2017). Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila. Sci Rep 7(1): 11368. PubMed ID: 28900300

    Koenig, S., Wolf, R. and Heisenberg, M. (2016). Visual attention in flies-dopamine in the mushroom bodies mediates the after-effect of cueing. PLoS One 11: e0161412. PubMed ID: 27571359

    Kuo, S. Y., Wu, C. L., Hsieh, M. Y., Lin, C. T., Wen, R. K., Chen, L. C., Chen, Y. H., Yu, Y. W., Wang, H. D., Su, Y. J., Lin, C. J., Yang, C. Y., Guan, H. Y., Wang, P. Y., Lan, T. H. and Fu, T. F. (2015). PPL2ab neurons restore sexual responses in aged Drosophila males through dopamine. Nat Commun 6: 7490. PubMed ID: 26123524

    Lebestky, T., et al. (2009). Two different forms of arousal in Drosophila are oppositely regulated by the dopamine D1 receptor ortholog DopR via distinct neural circuits. Neuron 64(4): 522-36. PubMed ID: 19945394

    Lewis, L.P., Siju, K.P., Aso, Y., Friedrich, A.B., Bulteel, A.J., Rubin, G.M. and Grunwald Kadow, I.C. (2015). A higher brain circuit for immediate integration of conflicting sensory information in Drosophila. Curr Biol 25(17):2203-14. PubMed ID: 26299514

    Li, Y., Hoffmann, J., Li, Y., Stephano, F., Bruchhaus, I., Fink, C. and Roeder, T. (2016). Octopamine controls starvation resistance, life span and metabolic traits in Drosophila. Sci Rep 6: 35359. PubMed ID: 27759117

    Liu, Q., Tabuchi, M., Liu, S., Kodama, L., Horiuchi, W., Daniels, J., Chiu, L., Baldoni, D. and Wu, M. N. (2017). Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger. Science 356(6337): 534-539. PubMed ID: 28473588

    Majdi, S., Berglund, E. C., Dunevall, J., Oleinick, A. I., Amatore, C., Krantz, D. E. and Ewing, A. G. (2015). Electrochemical measurements of optogenetically stimulated quantal amine release from single nerve cell varicosities in Drosophila larvae. Angew Chem Int Ed Engl [Epub ahead of print]. PubMed ID: 26387683

    Majeed, Z. R., Abdeljaber, E., Soveland, R., Cornwell, K., Bankemper, A., Koch, F. and Cooper, R. L. (2016). Modulatory action by the serotonergic system: behavior and neurophysiology in Drosophila melanogaster. Neural Plast 2016: 7291438. PubMed ID: 26989517

    Mednick, S. C., Cai, D. J., Shuman, T., Anagnostaras, S. and Wixted, J. T. (2011). An opportunistic theory of cellular and systems consolidation. Trends Neurosci 34: 504-514. PubMed ID: 21742389

    Nall, A.H., Shakhmantsir, I., Cichewicz, K., Birman, S., Hirsh, J. and Sehgal, A. (2016) Caffeine promotes wakefulness via dopamine signaling in Drosophila. Sci Rep 6: 20938. PubMed ID: 26868675

    Ohhara, Y., Shimada-Niwa, Y., Niwa, R., Kayashima, Y., Hayashi, Y., Akagi, K., Ueda, H., Yamakawa-Kobayashi, K. and Kobayashi, S. (2015). Autocrine regulation of ecdysone synthesis by β3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis. Proc Natl Acad Sci USA [Epub ahead of print]. PubMed ID: 25605909

    Owald, D., Felsenberg, J., Talbot, C. B., Das, G., Perisse, E., Huetteroth, W. and Waddell, S. (2015). Activity of defined mushroom body output neurons underlies learned olfactory behavior in Drosophila. Neuron 86: 417-427. PubMed ID: 25864636

    Pimentel, D., Donlea, J.M., Talbot, C.B., Song, S.M., Thurston, A.J. and Miesenbock, G. (2016). Operation of a homeostatic sleep switch. Nature 536(7616):333-7. PubMed ID: 27487216

    Pool, A. H., Kvello, P., Mann, K., Cheung, S. K., Gordon, M. D., Wang, L. and Scott, K. (2014). Four GABAergic interneurons impose feeding restraint in Drosophila. Neuron 83: 164-177. PubMed ID: 24991960

    Pooryasin, A. and Fiala, A. (2015). Identified serotonin-releasing neurons induce behavioral quiescence and suppress mating in Drosophila. J Neurosci 35: 12792-12812. PubMed ID: 26377467'

    Qi, C. and Lee, D. (2014). Pre- and postsynaptic role of Dopamine D2 Receptor DD2R in Drosophila olfactory associative learning. Biology (Basel) 3: 831-845. PubMed ID: 25422852

    Ren, Q., Awasaki, T., Huang, Y.F., Liu, Z. and Lee, T. (2016). Cell class-lineage analysis reveals sexually dimorphic lineage compositions in the Drosophila brain. Curr Biol 26(19):2583-2593. PubMed ID: 27618265

    Ries, A. S., Hermanns, T., Poeck, B. and Strauss, R. (2017). Serotonin modulates a depression-like state in Drosophila responsive to lithium treatment. Nat Commun 8: 15738. PubMed ID: 28585544

    Rohwedder, A., Wenz, N. L., Stehle, B., Huser, A., Yamagata, N., Zlatic, M., Truman, J. W., Tanimoto, H., Saumweber, T., Gerber, B. and Thum, A. S. (2016). Four individually identified paired dopamine neurons signal reward in larval Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 26877086

    Sadaf, S., Reddy, O. V., Sane, S. P. and Hasan, G. (2015). Neural control of wing coordination in flies. Curr Biol 25(1): 80-86. PubMed ID: 25496964

    Scholz-Kornehl, S. and Schwarzel, M. (2016). Circuit analysis of a Drosophila Dopamine type 2 receptor that supports anesthesia-resistant memory. J Neurosci 36: 7936-7945. PubMed ID: 27466338

    Schoofs, A., Huckesfeld, S. and Pankratz, M. J. (2017). Serotonergic network in the subesophageal zone modulates the motor pattern for food intake in Drosophila. J Insect Physiol [Epub ahead of print]. PubMed ID: 28735009

    Sealover, N. R., Felts, B., Kuntz, C. P., Jarrard, R. E., Hockerman, G. H., Barker, E. L. and Henry, L. K. (2016). The external gate of the human and Drosophila serotonin transporters requires a basic/acidic amino acid pair for MDMA translocation and the induction of substrate efflux. Biochem Pharmacol [Epub ahead of print]. PubMed ID: 27638414

    Shyu, W. H., Chiu, T. H., Chiang, M. H., Cheng, Y. C., Tsai, Y. L., Fu, T. F., Wu, T. and Wu, C. L. (2017). Neural circuits for long-term water-reward memory processing in thirsty Drosophila. Nat Commun 8: 15230. PubMed ID: 28504254

    Sitaraman, D., Aso, Y., Jin, X., Chen, N., Felix, M., Rubin, G. M. and Nitabach, M. N. (2015a). Propagation of homeostatic sleep signals by segregated synaptic microcircuits of the Drosophila mushroom body. Curr Biol 25: 2915-2927. PubMed ID: 26455303.

    Sitaraman, D., Aso, Y., Rubin, G.M. and Nitabach, M.N. (2015b). Control of sleep by dopaminergic inputs to the Drosophila mushroom body. Front Neural Circuits 9: 73. PubMed ID: 26617493

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

    Tao, J., Bulgari, D., Deitcher, D. L. and Levitan, E. S. (2017). Limited distal organelles and synaptic function in extensive monoaminergic innervation. J Cell Sci [Epub ahead of print]. PubMed ID: 28600320

    Ueno, T., et al. (2012). Identification of a dopamine pathway that regulates sleep and arousal in Drosophila. Nat. Neurosci. 15(11): 1516-23. PubMed ID: 23064381

    Vömel, M. and Wegener, C. (2008). Neuroarchitecture of aminergic systems in the larval ventral ganglion of Drosophila melanogaster. PLoS ONE 3(3): e1848. PubMed Citation: 18365004

    Yamagata, N., Hiroi, M., Kondo, S., Abe, A. and Tanimoto, H. (2016). Suppression of dopamine neurons mediates reward. PLoS Biol 14: e1002586. PubMed ID: 27997541

    Yang, Z., Yu, Y., Zhang, V., Tian, Y., Qi, W. and Wang, L. (2015). Octopamine mediates starvation-induced hyperactivity in adult Drosophila. Proc Natl Acad Sci U S A 112: 5219-5224. PubMed ID: 25848004

    Yu, Y., Huang, R., Ye, J., Zhang, V., Wu, C., Cheng, G., Jia, J. and Wang, L. (2016). Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila. Elife 5: e15693. PubMed ID: 27612383

    Xu, L., He, J., Kaiser, A., Graber, N., Schlager, L., Ritze, Y. and Scholz, H. (2016). A single pair of serotonergic neurons counteracts serotonergic inhibition of ethanol attraction in Drosophila. PLoS One 11(12): e0167518. PubMed ID: 27936023



    Zygotically transcribed genes

    Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

    The Interactive Fly resides on the
    Society for Developmental Biology's Web server.