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
  • Shared mushroom body circuits underlie visual and olfactory memories in Drosophila
  • Activity of defined mushroom body output neurons underlies learned olfactory behavior in Drosophila
  • Study of the release of endogenous amines in Drosophila brain in vivo in response to stimuli linked to aversive olfactory conditioning
  • Coordinated and compartmentalized neuromodulation shapes sensory processing in Drosophila
  • Re-evaluation of learned information in Drosophila
  • Novel role for ecdysone in Drosophila conditioned behavior: Linking GPCR-mediated non-canonical steroid action to cAMP signaling in the adult brain
  • 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
  • Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila
  • 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
  • A decision underlies phototaxis in an insect]
  • Cyclic AMP-dependent plasticity underlies rapid changes in odor coding associated with reward learning
  • The neural circuit linking mushroom body parallel circuits induces memory consolidation in Drosophila
  • A connectome of a learning and memory center in the adult Drosophila brain
  • Dopamine and octopamine differentiate between aversive and appetitive olfactory memories 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
  • Dopamine neurons modulate pheromone responses in Drosophila courtship learning
  • Layered reward signalling through octopamine and dopamine in Drosophila
  • Dopamine receptor DAMB signals via Gq to mediate forgetting in Drosophila
  • Four individually identified paired dopamine neurons signal reward in larval Drosophila
  • Reciprocal synapses between mushroom body and dopamine neurons form a positive feedback loop required for learning
  • Suppression of GABAergic neurons through D2-like receptor secures efficient conditioning in Drosophila aversive olfactory learning
  • Independent contributions of discrete dopaminergic circuits to cellular plasticity, memory strength, and valence in Drosophila
  • Reward signaling in a recurrent circuit of dopaminergic neurons and peptidergic Kenyon cells
  • Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics
  • A neural mechanism for deprivation state-specific expression of relevant memories in Drosophila
  • Dopaminergic neurons can influence heat-box place learning in Drosophila
  • Distinct dopamine receptor pathways underlie the temporal sensitivity of associative learning
  • Input Connectivity Reveals Additional Heterogeneity of Dopaminergic Reinforcement in Drosophila
  • Carbon monoxide, a retrograde messenger generated in postsynaptic mushroom body neurons, evokes noncanonical dopamine release
  • Serotonin receptor 5-HT7 in Drosophila mushroom body neurons mediates larval appetitive olfactory learning
  • Dopaminergic mechanism underlying reward-encoding of punishment omission during reversal learning in Drosophila
  • Associative learning in larval and adult Drosophila is impaired by the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine
  • Dopamine-based mechanism for transient forgetting ).

    Dopamine, Behavior, Addiction, Disease and Aging
  • Active and passive sexual roles that arise in Drosophila male-male courtship are modulated by dopamine levels in PPL2ab neurons
  • Contribution of DA signaling to appetitive odor perception in a Drosophila model
  • Circuits that encode and guide alcohol-associated preference
  • A feedforward circuit regulates action selection of pre-mating courtship behavior in female Drosophila
  • Transient Administration of Dopaminergic Precursor Causes Inheritable Overfeeding Behavior in Young Drosophila melanogaster Adults
  • Disruption of dopamine homeostasis has sexually dimorphic effects on senescence characteristics of Drosophila melanogaster
  • PPL2ab neurons restore sexual responses in aged Drosophila males through dopamine
  • Novel Drosophila model for parkinsonism by targeting phosphoglycerate kinase
  • Identification of a New Allosteric Binding Site for Cocaine in Dopamine Transporter
  • Multiple Pathways of LRRK2-G2019S/Rab10 Interaction in Dopaminergic Neurons

    Biogenic amines and Aggression
  • Aggressive behavior
  • Octopamine in male aggression of Drosophila
  • A subset of octopaminergic neurons are important for Drosophila aggression
  • Modulation of Drosophila male behavioral choice
  • Trace amines differentially regulate adult locomotor activity, cocaine sensitivity, and female fertility in Drosophila melanogaster
  • Trace conditioning in Drosophila induces associative plasticity in mushroom body kenyon cells and dopaminergic neurons
  • 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
  • Serotonergic modulation of aggression in Drosophila involves GABAergic and cholinergic opposing pathways
  • Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons

    Biogenic Amines, Appetite Control and Food Choice
  • Visualizing neuromodulation in vivo: TANGO-mapping of dopamine signaling reveals appetite control of sugar sensing
  • Dopaminergic modulation of sucrose acceptance behavior in Drosophila
  • A higher brain circuit for immediate integration of conflicting sensory information in Drosophila
  • Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger
  • 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
  • Unveiling the dual role of the dopaminergic system on locomotion and the innate value for an aversive olfactory stimulus in Drosophila
  • Recurrent architecture for adaptive regulation of learning in the insect brain

    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
  • Drosophila D1 dopamine receptor mediates caffeine-induced arousal
  • D1 receptor activation in the mushroom bodies rescues sleep-loss-induced learning impairments in Drosophila
  • Sleep facilitates memory by blocking dopamine neuron-mediated forgetting
  • Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila
  • 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
  • Visual attention in flies-dopamine in the mushroom bodies mediates the after-effect of cueing
  • Operation of a homeostatic sleep switch
  • Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila
  • LAT1-like transporters regulate dopaminergic transmission and sleep in Drosophila
  • Dopamine signaling in wake promoting clock neurons is not required for the normal regulation of sleep in Drosophila

    Octopamine functions as a neuromodulator: regulation of reward signaling, synaptic and behaviorial plasticity, activity, sleep, aggression, starvation resistance, appetite, metabolic traits, life span, ovulation
  • A comprehensive anatomical map of the peripheral octopaminergic/tyraminergic system of Drosophila melanogaster
  • Autoregulatory and paracrine control of synaptic and behavioral plasticity by octopaminergic signaling
  • Inhibitory control of synaptic and behavioral plasticity by octopaminergic signaling
  • Octopamine mediates starvation-induced hyperactivity in adult Drosophila
  • Octopamine drives endurance exercise adaptations in Drosophila
  • Octopamine controls starvation resistance, life span and metabolic traits in Drosophila
  • Identification of a neural circuit that underlies the effects of octopamine on sleep:wake behavior
  • Tyramine action on motoneuron excitability and adaptable tyramine/octopamine ratios adjust Drosophila locomotion to nutritional state
  • Octopamine-mediated circuit mechanism underlying controlled appetite for palatable food in Drosophila
  • Drosophila seminal protein Ovulin mediates ovulation through female octopamine neuronal signaling
  • Octopamine regulates sleep in Drosophila through protein kinase A-dependent mechanisms
  • Octopamine plays a critical role in HSPG glypican regulation of experience-dependent synaptic and behavioral plasticity by modulating the non-canonical BMP pathway
  • AANAT1 functions in astrocytes to regulate sleep homeostasis

    Serotonin functions as a neuromodulator: regulation of mating, aggression, sensory plasticity, hunger, food intake, addiction, sleep and memory
  • Pharmacological characterization of the fifth serotonin receptor subtype of Drosophila melanogaster
  • Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster
  • Dopamine and serotonin are both required for mate-copying in Drosophila melanogaster
  • Single serotonergic neurons that modulate aggression 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
  • A single pair of serotonergic neurons counteracts serotonergic inhibition of ethanol attraction in Drosophila
  • Serotonergic network in the subesophageal zone modulates the motor pattern for food intake in Drosophila
  • Discrete serotonin systems mediate memory enhancement and escape latencies after unpredicted aversive experience in Drosophila place memory
  • Characterization of a novel Drosophila SERT mutant: insights on the contribution of the serotonin neural system to behaviors
  • Identified serotonin-releasing neurons induce behavioral quiescence and suppress mating in Drosophila
  • 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
  • Serotonin modulates a depression-like state in Drosophila responsive to lithium treatment
  • A serotonin-modulated circuit controls sleep architecture to regulate cognitive function independent of total sleep in Drosophila
  • Serotonergic modulation of visual neurons in Drosophila melanogaster
  • A conserved role for sleep in supporting Spatial Learning in Drosophila
  • Serotoninergic Modulation of Phototactic Variability Underpins a Bet-Hedging Strategy in Drosophila melanogaster

    Biogenic Amines, Development and Physiology
  • Developmental analysis of the dopamine-containing neurons of the Drosophila brain
  • 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
  • Aminergic signaling controls ovarian dormancy in Drosophila
  • A population of interneurons signals changes in the basal concentration of serotonin and mediates gain control in the Drosophila antennal lobe
  • Neurochemical organization of the Drosophila brain visualized by endogenously tagged neurotransmitter receptors
  • Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission
  • Bidirectional regulation of glial potassium buffering: glioprotection versus neuroprotection
  • Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octbeta2R cascade

    Biogenic Amines and Stress
  • Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation
  • Enabling cell-type-specific behavioral epigenetics in Drosophila: a modified high-yield INTACT method reveals the impact of social environment on the epigenetic landscape in dopaminergic neurons
  • gamma-Oryzanol produces an antidepressant-like effect in a chronic unpredictable mild stress model of depression in Drosophila melanogaster
  • Iron overload during the embryonic period develops hyperactive like behavior and dysregulation of biogenic amines in Drosophila melanogaster

  • Genes involved in synthesis of biogenic amines

  • Genes regulating the synthesis and function of biogenic amines

    A comprehensive anatomical map of the peripheral octopaminergic/tyraminergic system of Drosophila melanogaster

    The modulation of an animal's behavior through external sensory stimuli, previous experience and its internal state is crucial to survive in a constantly changing environment. In most insects, octopamine (OA) and its precursor tyramine (TA) modulate a variety of physiological processes and behaviors by shifting the organism from a relaxed or dormant condition to a responsive, excited and alerted state. Even though OA/TA neurons of the central brain are described on single cell level in Drosophila melanogaster, the periphery was largely omitted from anatomical studies. Given that OA/TA is involved in behaviors like feeding, flying and locomotion, which highly depend on a variety of peripheral organs, it is necessary to study the peripheral connections of these neurons to get a complete picture of the OA/TA circuitry. This study describes the anatomy of this aminergic system in relation to peripheral tissues of the entire fly. OA/TA neurons arborize onto skeletal muscles all over the body and innervate reproductive organs, the heart, the corpora allata, and sensory organs in the antennae, legs, wings and halteres underlining their relevance in modulating complex behaviors (Pauls, 2018).

    The adrenergic system of mammals influences various aspects of the animal's life. Its transmitters/hormones, adrenaline and noradrenaline, modulate a variety of physiological processes and behaviors. They are secreted into the bloodstream by the adrenal glands in response to stress. In addition, they are synthesized and released by axonal terminals in the central nervous system (CNS) as well as sympathetic fibers of the autonomic nervous system. Adrenaline and noradrenaline have been described as modulators to shift the organism from a relaxed or dormant state to a responsive, excited and alerted state. Stressful stimuli induce a metabolic and behavioral adaptation, leading to enhanced energy supply, increased muscle performance, increased sensory perception and a matched behavior. This so-called 'fight or flight' response can be seen in vertebrates and invertebrates. In insects, the stress response is mediated -- among others -- by octopamine (OA) and its precursor tyramine (TA). TA is synthesized from tyrosine by the action of a tyrosine decarboxylase enzyme (Tdc) and functions as an independent neurotransmitter/-modulator as well as the intermediate step in OA synthesis. For this, TA is catalyzed by the tyramine-β-hydroxylase (TΒH). Similar to the vertebrate adrenergic system, OA and TA act through specific G-protein coupled receptors. Besides structural similarities between OA/TA and adrenaline/noradrenaline and the corresponding receptors, functional similarities are illustrated by the action of these transmitters/hormones in the regulation of physiological processes and behaviors. OA and TA are known to modulate muscle performance, glycogenolysis, fat metabolism, heart rate, and respiration in insects (Pauls, 2018).

    While the role of TA as an independent signaling molecule was underestimated for a long time, OA has been extensively studied and was shown to have effects on almost every organ, sensory modality and behavior in a great variety of insects. The most intensively studied peripheral organs regarding the modulatory role of OA are muscles. Here, OA is thought to not exclusively modulate muscle performance or motor activity. OA rather modulates muscle action according to metabolic and physiological processes, for example by promoting energy mobilization directly from the fat body, or indirectly by promoting the release of adipokinetic homones (AKH) from neuroendocrine cells in the corpora cardiaca (CC, a homolog of the vertebrate anterior pituitary gland and an analog of mammalian pancreatic alpha cells). In addition to the impact of OA/TA on muscles, fat body and AKH cells, OA is shown to modulate the heart, trachea and air sacs, gut, hemocytes, salivary glands, Malpighian tubules and ovaries in insects, mainly to induce a general stress or arousal state. However, in total OA seems to modulate a vast number of behaviors, which are not necessarily coupled to stress responses. The OA/TA system is shown to also act on inter alia (i.a.) learning and memory, sleep, feeding, flight, locomotion, and aggression (Pauls, 2018).

    As mentioned above, OA and TA act as neurotransmitters and neuromodulators, allowing them to act in a paracrine, endocrine or autocrine fashion. In the fruit fly Drosophila, huge efforts were made to describe OA/TA neurons (OANs/TANs) in the brain and ventral nervous system (VNS) down to the single cell level. Nevertheless, although knowledge about physiological processes and behaviors modulated by the OA/TA system in the brain is rich, less is known about how OA and TA reach all its target organs and tissues in the periphery (exceptions: reproductive organs and muscles) (Pauls, 2018).

    This study used the genetically tractable fruit fly Drosophila melanogaster to describe the arborizations of Tdc2-Gal4-positive, and therefore OANs and TANs in the periphery, as the Drosophila Tdc2 gene is expressed neurally. OANs/TANs were found to be widespread distributed throughout the fly's body with innervations in the skeletal muscles, reproductive organs, corpora allata, antennae, legs, wings, halteres and the heart. This diverse innervation pattern reflects the modulatory role of OA/TA in many different behaviors and physiological processes. These results provide, for the very first time, a complete and comprehensive map of the OA/TA circuitry in the entire insect body. This map allows assumptions about the type of OA/TA signaling (paracrine or endocrine) to a specific organ and, at the same time, it provides a deeper understanding to what extend the OA/TA-dependent activity of peripheral organs is altered, for example by genetically manipulating Tdc2-Gal4-positive neurons in the brain and VNS (Pauls, 2018).

    This study used the Tdc2-Gal4 line, allowing Gal4 expression under the control of a regulatory sequence of the tyrosine decarboxylase enzyme. As this enzyme is essential for the synthesis of TA from tyrosine, the Tdc2-Gal4-line labels both TANs and OANs. Within the Drosophila brain, Tdc2-Gal4 labels in total about 137 cells, while additional 39 cells are located in the VNS. The small number of Tdc2Ns lead to arborizations in large parts of the central brain, optic lobes and the thoracic and abdominal ganglion. Based on the profound innervation of Tdc2Ns in the brain and VNS, the variety of behaviors modulated by the OA/TA system including learning and memory, feeding, vision, and sleep, are not surprising. Beyond the brain and VNS, OANs and TANs massively innervate regions within the periphery of the fly. This study described arborizations on most skeletal muscles, the antennae, wings, halteres and reproductive system and parts of the circulatory system and stomodaeal ganglion (Pauls, 2018).

    The findings are in line with previous reports focusing on the expression of different OA and TA receptors in the fly. Accordingly, the OA receptor OAMB is expressed in reproductive organs (in both male and female flies) and muscles, which are directly innervated by Tdc2Ns. Additionally, the midgut and trachea contain OA and TA receptors, but do not seem to be innervated by Tdc2Ns, even though axons run in close vicinity to these organs. Likewise, the OA receptor Octβ2R is expressed in the fat body, salivary glands and Malpighian tubules, tissues that seem not to be innervated by Tdc2Ns, while the expression of Octβ1R and Octβ3R is more specific56,57. The three tyramine receptors TyrR, TyrRII and TyrRIII show a broad expression in the periphery, also in tissues not innervated by Tdc2Ns56. The lack of direct innervation of these peripheral tissues might argue for volume transmission or hemolymph released OA/TA from Tdc2Ns. Alternatively, Drosophila TDC1, the product of the non-neurally expressed Tdc gene, is expressed in the gut musculature, rectal papillae, Malpighian tubules and two small clusters in the thoracic nervous system and might be the source for peripheral TA. Interestingly, TyrR seems to be the only receptor expressed in the heart, suggesting that only TA modulates heart function. Contrary, OA has a modulatory effect on the heart of other insect species including honeybees, olive fruit flies and cockroaches. This is also in line with a previous report providing evidence that OA modulates the heart rate of the Drosophila fly and pupa, but not the larva (Pauls, 2018).

    OA-dependent modulation of organs and tissues is mainly elicited through muscle action, especially in terms of its impact on the 'fight or flight' response. In line with this, it was observed Tdc2-Gal4-positive arborizations on nearly all skeletal muscles and many visceral muscles. In both Drosophila and desert locusts OA and TA is expressed in type II terminals of skeletal muscles50. OA has an excitatory effect on Drosophila flight muscles, while TA was shown to inhibit excitatory junction potentials, and thereby reduce muscle contractions and locomotion at least in the larva. In addition, flies lacking OA show severe deficits in flight initiation and maintenance. Interestingly, in an antagonistic effect to serotonin, OA reduces crop muscle activity presumably via Octβ1R, suggesting that OA has different effects on muscle activity dependent on the type of muscle. However, the data do not provide any convincing evidence of a direct innervation of Tdc2Ns of the crop, even though many fibers run in close vicinity, suggesting that OA might target the crop by volume transmission (Pauls, 2018).

    Furthermore, OA modulates ovulation and fertilization in insects. Flies lacking OA display a severe egg-laying phenotype. Remarkably, within the female reproductive organ two different OA receptors, OAMB and Octβ2R, are necessary. Again, OA has a strong impact on muscle activity within the reproductive system. Octβ2R is expressed in the visceral oviduct muscle and elicits muscle relaxation through an increase of intracellular cAMP levels. Such an OA-dependent modulation appears to be conserved as OA is found in dorsal unpaired median neurons of locusts innervating oviduct muscles through the oviducal nerve. However, the data suggest that OA-positive fibers not only innervate oviduct muscles, but also enter the organs themselves. The OAMB receptor is expressed in epithelial cells inducing fluid secretion through increasing intracellular Ca2+ levels. Thus, OA affects different processes within the female reproductive organ due to the expression of different receptors and their coupled signaling pathways, which may be a general mechanism of the OA/TA system to fulfill an extensive modulatory function (Pauls, 2018).

    OA does not exclusively modulate muscle activity, but also sensory neurons of external tissues like the antennae, halteres and wings. OA has also been shown to increase the spontaneous activity of olfactory receptor neurons (ORN). The modulation of ORNs allows OA to modulate the innate response to attractive stimuli like fruit odors or pheromones. Further, this modulation helps nestmate recognition in ants. In addition to Tdc2-Gal4-positive arborizations in the funiculus, Tdc2-Gal4-positive sensory neurons were found in the JO, a chordotonal organ sensitive to mechanosensory stimuli and thus important for hearing in insects. In mosquitos, OA modulates auditory frequency tuning and thereby affects mating behavior. In locusts, OA similarly modulates the response of chordotonal neurons in the legs to encode proprioceptive information. These data suggest that chordotonal neurons in the legs, wings, halteres and thorax are included in the Tdc2-Gal4 line suggesting a conserved modulatory role of OA/TA for insect proprioception (Pauls, 2018).

    Taken together, this study suggests that the OA/TA system massively modulates various organs and tissues in the periphery of Drosophila. Through distinct receptors and coupled signaling pathways OANs/TANs mainly induce 'fight or flight' responses by modulating muscle activity, proprioception, and heart rate. As a result, the innervation pattern in the periphery supports the idea that the OA/TA system is crucial for insects to switch from a dormant to an excited state, by a positive modulation of muscle activity, heart rate and energy supply, and a simultaneous negative modulation of physiological processes like sleep (Pauls, 2018).

    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).

    Shared mushroom body circuits underlie visual and olfactory memories in Drosophila

    In nature, animals form memories associating reward or punishment with stimuli from different sensory modalities, such as smells and colors. It is unclear, however, how distinct sensory memories are processed in the brain. This study established appetitive and aversive visual learning assays for Drosophila that are comparable to the widely used olfactory learning assays. These assays share critical features, such as reinforcing stimuli (sugar reward and electric shock punishment), and allow direct comparison of the cellular requirements for visual and olfactory memories. It was found that the same subsets of dopamine neurons drive formation of both sensory memories. Furthermore, distinct yet partially overlapping subsets of mushroom body intrinsic neurons are required for visual and olfactory memories. Thus, these results suggest that distinct sensory memories are processed in a common brain center. Such centralization of related brain functions is an economical design that avoids the repetition of similar circuit motifs (Vogt, 2014).

    Devising a transparent electric shock grid module made it possible to apply the same visual stimulation in aversive and appetitive conditioning assays. Also an integrated platform was developed for fully automated high-throughput data acquisition using customized software to control the presentation of electric shock and visual stimuli while making video recordings of behavior. In these assays, memory performance is based on altered visual preference in walking flies, a task likely to be less demanding than the constant flight required for flight simulator learning. These advantages facilitate behavioral examination of many genotypes (Vogt, 2014).

    Circuits underlying olfactory and visual memory can be optimally compared when the sugar reward and electric shock punishment are matched between the two modalities. Visual and olfactory memories share the same subsets of dopamine neurons that convey reinforcing signals. This shared requirement of the transmitter system between visual and olfactory learning has been described in crickets. However, the pharmacological manipulation used in these studies does not allow further circuit dissection (Vogt, 2014).

    For electric shock reinforcement, identified neurons in the PPL1 cluster, such as MB-MP1, MB-MV1 and MB-V1, drive aversive memories in both visual and olfactory learning, while the MB-M3 neurons in the PAM cluster seem to be involved specifically in aversive olfactory memory. Thus, overlapping sets of dopamine neurons appear to represent electric shock punishment in both visual and olfactory learning with olfactory aversive memory probably recruiting a larger set. Previous studies have shown that the MB-M3 neurons induce aversive olfactory memory that increases stability of other memory components. Olfactory memories last longer than visual memories potentially due to the recruitment of additional dopamine neurons (Vogt, 2014).

    In appetitive conditioning, PAM cluster neurons play crucial roles in both olfactory and visual memories. Which cell types in these clusters are involved and whether there is a cellular distinction between olfactory and visual memory requires further analysis at the single cell level. Most importantly, all these neurons convey dopamine signals to restricted subdomains of the MB. The blockade of octopamine neurons did not impair appetitive visual memories with sucrose. The involvement of octopamine neurons may be more substantial when non-nutritious sweet taste rewards are used, as has been shown in olfactory learning (Vogt, 2014).

    In addition to these shared reinforcement circuits in the MB, the necessity of MB output for visual memory acquisition and retrieval is also consistent with olfactory conditioning. Taken together, these results suggest that the MBs harbor associative plasticity for visual memories and support the conclusion that similar coincidence detection mechanisms are used to form memories within the MBs. Centralization of similar brain functions spares the cost of maintaining similar circuit motifs in different brain areas and may be an evolutionary conserved design of information processing. Such converging inputs of different stimuli into a multisensory area have even been described in humans (Vogt, 2014).

    'Flight simulator' visual learning was shown to require the central complex but not the MBs. Although this appears to contradict the current study, it is noted that there are important differences between the behavioral paradigms employed. In the flight simulator, a single tethered flyingDrosophilais trained to associate a specific visual cue with a laser beam punishment, to later on avoid flying towards this cue in the test. Although this study controlled for visual context consistency and the 'operant component' of the flight simulator training, any other difference could account for the differential requirement of brain structures. Given that flies during flight show octopamine-mediated modulation of neurons in the optic lobe, similar state-dependent mechanisms might underlie different requirement of higher brain centers. Thus, it is critical to design comparable memory paradigms (Vogt, 2014).

    This study together with previous results in associative taste learning highlights the fact that the role of the MB in associative learning is not restricted to one sensory modality or reinforcer. This study found that olfactory and visual memories recruit overlapping, yet partly distinct, sets of Kenyon cells. In contrast to the well-described olfactory projection neurons, visual inputs to the MB remain unidentified. No anatomical evidence has been reported in Drosophila for direct connections between optic lobes and MBs although such connections are found in other insects. Also afferents originating in the protocerebrum were found to provide multi-modal input to the MB lobes of cockroaches. Thus, Drosophila MBs may receive indirect visual input from optic lobes, and the identification of such a visual pathway would significantly contribute to understanding of the MB circuits (Vogt, 2014).

    Given the general requirement of the γ lobe neurons, visual and olfactory cues may be both represented in the γ neurons. Consistently, the dopamine neurons that convey appetitive and aversive memories heavily project to the γ lobe. In olfactory conditioning, the γ lobe was shown to contribute mainly to short-term memory. This converging evidence from olfactory and visual memories suggests a general role for the γ lobe in short-lasting memories across different sensory modalities. Previous studies found that the MB is also involved in sensorimotor gating of visual stimuli or visual selective attention. Therefore, the MB circuits for visual associative memories might be required for sensorimotor gating and attention (Vogt, 2014).

    Interestingly, the contribution of the α'/β' lobes is selective for olfactory memories. This Kenyon cell class is more specialized to odor representation, as the cells have the broadest odor tuning and the lowest response threshold among the three Kenyon cell types (Vogt, 2014).

    The role of α/β neurons in visual memories is also limited. The α/β neurons might play more modulatory roles in specific visual memory tasks, such as context generalization, facilitation of operant learning and occasion setting. This modulatory role of the α/β neurons is corroborated in olfactory learning, where they are preferentially required for long-lasting memories (Vogt, 2014).

    Differentiated but overlapping sensory representations by KCs may be conserved among insect species. In honeybees, different sensory modalities are represented in spatially segregated areas of the calyx, whereas the basal ring region receives visual and olfactory inputs. The MB might thus have evolved to represent the sensory space of those modalities that are subject to associative modulation (Vogt, 2014).

    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 drives endurance exercise adaptations in Drosophila

    Endurance exercise is an effective therapeutic intervention with substantial pro-healthspan effects. Male Drosophila respond to a ramped daily program of exercise by inducing conserved physiological responses similar to those seen in mice and humans. Female flies respond to an exercise stimulus but do not experience the adaptive training response seen in males. This study used female flies as a model to demonstrate that differences in exercise response are mediated by differences in neuronal activity. The activity of octopaminergic neurons is specifically required to induce the conserved cellular and physiological changes seen following endurance training. Furthermore, either intermittent, scheduled activation of octopaminergic neurons or octopamine feeding is able to fully substitute for exercise, conferring a suite of pro-healthspan benefits to sedentary Drosophila. These experiments indicate that octopamine is a critical mediator of adaptation to endurance exercise in Drosophila (Sujkowski, 2017).

    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).

    Novel Drosophila model for parkinsonism by targeting phosphoglycerate kinase

    Patients with Parkinson's disease (PD) show a common progressive neurodegenerative movement disorder characterized by rigidity, tremors, postural instability, and bradykinesia due to the loss of dopaminergic neurons in the substantia nigra, and is often accompanied by several non-motor symptoms, called parkinsonism. Several lines of recent evidence support the hypothesis that mutations in the gene encoding phosphoglycerate kinase (PGK) play an important role in the PD mechanism. PGK is a key enzyme in the glycolytic pathway that catalyzes the reaction from 1,3-diphosphoglycerate to 3-phosphoglycerate. This study established a parkinsonism model targeting Drosophila Pgk. Dopaminergic (DA) neuron-specific Pgk knockdown lead to locomotive defects in both young and aged adult flies and was accompanied by progressive DA neuron loss with aging. Pgk knockdown in DA neurons decreased dopamine levels in the central nervous system (CNS) of both young and aged adult flies. These phenotypes are similar to the defects observed in human PD patients, suggesting that the Pgk knockdown flies established herein are a promising model for parkinsonism. Furthermore, pan-neuron-specific Pgk knockdown induced low ATP levels and the accumulation of reactive oxygen species (ROS) in the CNS of third instar larvae. Collectively, these results indicate that a failure in the energy production system of Pgk knockdown flies causes locomotive defects accompanied by neuronal dysfunction and degeneration in DA neurons (Shimizu, 2020).

    Identification of a New Allosteric Binding Site for Cocaine in Dopamine Transporter

    Dopamine (DA) transporter (DAT) is a major target for psychostimulant drugs of abuse such as cocaine that competitively binds to DAT, inhibits DA reuptake, and consequently increases synaptic DA levels. In addition to the central binding site inside DAT, the available experimental evidence suggests the existence of alternative binding sites on DAT, but detection and characterization of these sites are challenging by experiments alone. This study integrated multiple computational approaches to probe the potential binding sites on the wild-type Drosophila melanogaster DAT and identified a new allosteric site that displays high affinity for cocaine. This site is located on the surface of DAT, and binding of cocaine is primarily dominated by interactions with hydrophobic residues surrounding the site. Cocaine binding to this new site allosterically reduces the binding of DA/cocaine to the central binding pocket, and simultaneous binding of two cocaine molecules to a single DAT seems infeasible. Furthermore, binding of cocaine to this site was found to stabilize the conformation of DAT but alters the conformational population and thereby reduces the accessibility by DA, providing molecular insights into the inhibitory mechanism of cocaine. In addition, the results indicate that the conformations induced by cocaine binding to this site may be relevant to the oligomerization of DAT, highlighting a potential role of this new site in modulating the function of DAT (Xu, 2020).

    Multiple Pathways of LRRK2-G2019S/Rab10 Interaction in Dopaminergic Neurons

    Inherited mutations in the LRRK2 protein are the common causes of Parkinson's disease, but the mechanisms by which increased kinase activity of mutant LRRK2 leads to pathological events remain to be determined. In vitro assays (heterologous cell culture, phospho-protein mass spectrometry) suggest that several Rab proteins might be directly phosphorylated by LRRK2-G2019S. An in vivo screen of Rab expression in dopaminergic neurons in young adult Drosophila demonstrated a strong genetic interaction between LRRK2-G2019S and Rab10. To determine if Rab10 is necessary for LRRK2-induced pathophysiological responses in the neurons that control movement, vision, circadian activity, and memory. These four systems were chosen because they are modulated by dopaminergic neurons in both humans and flies. LRRK2-G2019S was expressed in Drosophila dopaminergic neurons and the effects of Rab10 depletion on Proboscis Extension, retinal neurophysiology, circadian activity pattern ('sleep'), and courtship memory determined in aged flies. Rab10 loss-of-function rescued LRRK2-G2019S induced bradykinesia and retinal signaling deficits. Rab10 knock-down, however, did not rescue the marked sleep phenotype which results from dopaminergic LRRK2-G2019S. Courtship memory is not affected by LRRK2, but is markedly improved by Rab10 depletion. Anatomically, both LRRK2-G2019S and Rab10 are seen in the cytoplasm and at the synaptic endings of dopaminergic neurons. It is concluded that, in Drosophila dopaminergic neurons, Rab10 is involved in some, but not all, LRRK2-induced behavioral deficits. Therefore, variations in Rab expression may contribute to susceptibility of different dopaminergic nuclei to neurodegeneration seen in people with Parkinson's disease (Fellgett, 2021).

    Contribution of DA signaling to appetitive odor perception in a Drosophila model

    Understanding cognitive processes that translate chemically diverse olfactory stimuli to specific appetitive drives remains challenging. Food-related odors arouse impulsive-like feeding of food media that are palatable and readily accessible in well-nourished Drosophila larvae. This study provides evidence that two assemblies of four dopamine (DA) neurons, one per brain hemisphere, contribute to perceptual processing of the qualitative and quantitative attributes of food scents. These DA neurons receive neural representations of chemically diverse food-related odors, and their combined neuronal activities become increasingly important as the chemical complexity of an appetizing odor stimulus increases. Furthermore, in each assembly of DA neurons, integrated odor signals are transformed to one-dimensional DA outputs that have no intrinsic reward values. Finally, a genetic analysis has revealed a D1-type DA receptor (Dop1R1)-gated mechanism in neuropeptide Y-like neurons that assigns appetitive significance to selected DA outputs. These findings suggest that fly larvae provide a useful platform for elucidation of molecular and circuit mechanisms underlying cognitive processing of olfactory and possibly other sensory cues (Pu, 2018).

    Neuronal reactivation during post-learning sleep consolidates long-term memory in Drosophila

    Animals consolidate some, but not all, learning experiences into long-term memory. Across the animal kingdom, sleep has been found to have a beneficial effect on the consolidation of recently formed memories into long-term storage. However, the underlying mechanisms of sleep dependent memory consolidation are poorly understood. This study shows that consolidation of courtship long-term memory in Drosophila is mediated by reactivation during sleep of dopaminergic neurons that were earlier involved in memory acquisition. Specific fan-shaped body neurons were identified that induce sleep after the learning experience and activate dopaminergic neurons for memory consolidation. Thus, this study provide a direct link between sleep, neuronal reactivation of dopaminergic neurons, and memory consolidation (Dag, 2019).

    The activity of DAN-aSP13s, which is essential for courtship memory acquisition, is also necessary during a discrete post-learning time window for LTM consolidation. Because neuronal reactivation occurs during sleep in rodents, it was hypothesized that post-learning activation of DAN-aSP13s involves a sleep-dependent mechanism. Using behavioral analysis and neuronal activity monitoring and perturbation approaches, thus study shows that DAN-aSP13s display an increased activity in freely behaving animals during sleep after a prolonged learning experience. This sleep is necessary for LTM consolidation, and it can be mediated by a specific class of sleep promoting neurons in the ventral layer of the fan shaped body (vFB). These vFB neurons consolidate courtship LTM in a discrete time window and provide an excitatory input to DAN-aSP13s. Thus, these data provide a causal link between sleep promoting neurons in the vFB, post-learning activation of dopaminergic neurons, and LTM consolidation (Dag, 2019).

    Based on these data, the following model is proposed for sleep-dependent consolidation of courtship LTM in Drosophila (see Post-learning activation of DAN-aSP13 neurons mediates LTM consolidation). During a prolonged learning experience, γKCs and DAN-aSP13s are repeatedly activated by olfactory and behavioral cues presented by an unreceptive female, respectively. Whereas prolonged wakefulness leads to an increase in homeostatic sleep drive in ellipsoid body neurons that in turn is conveyed to dFB, it is hypothesized that an extended learning experience generates a learning-dependent sleep drive that is transmitted to vFB. In turn, the vFB neurons enhance sleep after learning and provide an excitatory input back on DAN-aSP13s. It is thought that one potential site of a learning-dependent sleep drive are the MB neurons since they have been implicated in both memory formation and sleep regulation. Dopamine released upon DAN-aSP13 reactivation stimulates molecular processes in γKCs that are different from those engaged during STM acquisition and involve protein synthesis that is essential for LTM formation and persistence (Dag, 2019).

    Dopaminergic pathways are thought to convey information about whether an experience is rewarding or punishing and thus, worth remembering. Post-learning neuronal activity of the dopaminergic hippocampal inputs from the Ventral Tagmental Area (VTA) has been implicated in the consolidation of fear memory in rodents. Interestingly, this activity is critical during a discrete time window after learning. Post-learning activity of the VTA dopamine neurons has been also implicated in the reactivation during sleep of the hippocampal cells involved earlier in encoding of the spatial experience. This study shows that post-learning activation of DAN-aSP13s mediates the consolidation of courtship LTM in Drosophila. It is proposed that reactivation during sleep of the dopamine neurons that were previously active during memory acquisition ensures that spurious experiences are not admitted into LTM storage and thus only experiences that are either sufficiently salient or persistent become long-lasting memories. Specifically, reactivation of DAN-aSP13s during post-learning sleep enhances reactivation of the γKCs and cognate MBON-M6, which together with DAN-aSP13s form a recurrent circuit necessary for courtship memory acquisition. This study considers two hypotheses to account for this selectivity. During sleep, vFB neurons might selectively reactivate only the relevant DANs, or alternatively, they might activate all DANs but only the relevant subset is able to consolidate LTM. The selective-reactivation model would require some marker to distinguish which DANs were activated, whereas the selective-consolidation model would require a marker in the synapses of the γKCs that were earlier active during memory acquisition, for example translational regulator Orb2, which regulates translation upon neuronal activity during LTM consolidation (Dag, 2019).

    Activity of dopaminergic neurons regulates sleep-wake states in animals, including flies. Artificial activation of DAN-aSP13s has been shown to increase wakefulness. In contrast, the data presented in this study imply that activation of vFB neurons, although they activate DAN-aSP13s, do not promote wakefulness. These results suggest that activation of DAN-aSP13s by vFB neurons during sleep is qualitatively different from a direct optogenetic or thermogenetic activation used in previous studies. One potential explanation is that post-learning sleep involves activation of the vFB circuit, which provides both excitatory stimulus to DAN-aSP13s and inhibitory input to motor neurons. Another possibility is that post-learning sleep activates a subset od DAN-aSP13s that do not affect wakefulness (Dag, 2019).

    Thia study has identified a class of sleep-promoting neurons in the ventral layer of FB that are distinct from the well-studied sleep-promoting neurons in the dorsal layer of FB, which regulate sleep homeostasis. Given that vFB neurons enhance sleep and activate DAN-aSP13s for LTM consolidation, whereas dFB neurons are neither necessary nor sufficient for LTM consolidation, it is hypothesized that dFB and vFB neurons promote distinct components of sleep that have different functions. Homeostatic sleep is thought to facilitate memory encoding by downscaling synaptic weights and clearing metabolites from the brain accumulated during wakefulness. In contrast, the function of learning-dependent sleep might be to facilitate memory consolidation by strengthening synaptic connections that were engaged earlier during memory acquisition. Thus, the co-operation of homeostatic- and experience-dependent sleep would facilitate optimal conditions for learning new information and, if appropriate, incorporating it into long-term storage (Dag, 2019).

    Recent studies have implied that sleep in flies, as in humans and rodents, exhibits sleep stages characterized by distinct electrophysiological signatures. Interestingly, the signature of sleep that is induced by activation of the dFB neurons seems to have a simpler oscillatory pattern, recorded by local field potentials, than sleep that is induced by activation of the FB neurons comprising both dFB and vFB neurons. Thus, these data support the hypothesis that dFB and vFB neurons promote sleep with different properties and likely different functions (Dag, 2019).

    It is thought that sleep evolved in animals that are capable of complex learning which requires selective attention. Courtship learning is a multisensory form of learning that requires selective attention of a male to associate multiple learning cues presented by the mated female with the outcome of his own behavior. Accordingly, studies in bees have shown that sleep affects a complex form of learning such as spatial memory but has no role in the simple learning paradigm of proboscis extension. Hence, it would be interesting to investigate whether post-learning sleep is involved in the consolidation of other types of memory in Drosophila, such as the well-studied Pavlovian olfactory associative learning whereby animals associate an individual learning cue with a behavioral contingency (Dag, 2019).

    This work has established a functional link between a novel class of sleep-promoting neurons in the FB, post-learning reactivation of dopaminergic neurons and consolidation of courtship LTM. Moreover, the data suggest that sleep promoting vFB neurons mediate a learning-dependent regulation of sleep that is distinct from the homeostatic control which is facilitated by dFB neurons. Thus, this study uncovered a causal link between sleep-mediated neuronal reactivation and LTM consolidation in Drosophila. In addition, courtship LTM was establish in Drosophila as a tractable model to investigate the mechanisms that link learning-dependent sleep, neuronal reactivation and LTM consolidation (Dag, 2019).

    Suppression of GABAergic neurons through D2-like receptor secures efficient conditioning in Drosophila aversive olfactory learning

    The GABAergic system serves as a vital negative modulator in cognitive functions, such as learning and memory, while the mechanisms governing this inhibitory system remain to be elucidated. In Drosophila, the GABAergic anterior paired lateral (APL) neurons mediate a negative feedback essential for odor discrimination; however, their activity is suppressed by learning via unknown mechanisms. In aversive olfactory learning, a group of dopaminergic (DA) neurons is activated on electric shock (ES) and modulates the Kenyon cells (KCs) in the mushroom body, the center of olfactory learning. This study found that the same group of DA neurons also form functional synaptic connections with the APL neurons, thereby emitting a suppressive signal to the latter through Drosophila dopamine 2-like receptor (DD2R). Knockdown of either DD2R or its downstream molecules in the APL neurons results in impaired olfactory learning at the behavioral level. Results obtained from in vivo functional imaging experiments indicate that this DD2R-dependent DA-to-APL suppression occurs during odor-ES conditioning and discharges the GABAergic inhibition on the KCs specific to the conditioned odor. Moreover, the decrease in odor response of the APL neurons persists to the postconditioning phase, and this change is also absent in DD2R knockdown flies. Taken together, these findings show that DA-to-GABA suppression is essential for restraining the GABAergic inhibition during conditioning, as well as for inducing synaptic modification in this learning circuit. Such circuit mechanisms may play conserved roles in associative learning across species (Zhou, 2019).

    Independent contributions of discrete dopaminergic circuits to cellular plasticity, memory strength, and valence in Drosophila

    Dopaminergic neurons play a key role in encoding associative memories, but little is known about how these circuits modulate memory strength. This study reports that different sets of dopaminergic neurons projecting to the Drosophila mushroom body (MB) differentially regulate valence and memory strength. PPL2 neurons increase odor-evoked calcium responses to a paired odor in the MB and enhance behavioral memory strength when activated during olfactory classical conditioning. When paired with odor alone, they increase MB responses to the paired odor but do not drive behavioral approach or avoidance, suggesting that they increase the salience of the odor without encoding strong valence. This contrasts with the role of dopaminergic PPL1 neurons, which drive behavioral reinforcement but do not alter odor-evoked calcium responses in the MB when stimulated. These data suggest that different sets of dopaminergic neurons modulate olfactory valence and memory strength via independent actions on a memory-encoding brain region (Boto, 2019).

    Dopaminergic neurons are involved in associative learning across taxa. In Drosophila, activation of certain dopaminergic neurons during associative learning tasks drives conditioned approach or avoidance, suggesting that they function as part of the reinforcement pathway and may encode stimulus valence (positive or negative). There are eight clusters of dopaminergic neurons in the fly brain. Neurons in three clusters project to the mushroom body (MB), a region that receives olfactory information and is required for olfactory learning. PAM dopaminergic neurons project to the horizontal lobes (β, β', and γ); PPL1 neurons project to the vertical lobes (α and α'), heel, and peduncle; and PPL2ab neurons project to the calyx. Different subsets of PPL1 and PAM neurons modulate reinforcement during learning (Boto, 2019).

    Different sets of dopaminergic neurons play discrete roles in reinforcement during learning. Activating PPL1 dopaminergic neurons in lieu of reinforcement induces behavioral aversion to a paired odor. Conversely, activation of PAM neurons is sufficient to generate appetitive memories. These dopaminergic neurons respond strongly to the unconditioned stimulus during conditioning, releasing dopamine into the MB that integrates with odor-evoked spiking activity to drive learning-induced, cyclic AMP (cAMP)-dependent plasticity in the MB. Little is known about the third MB-innervating cluster, PPL2ab. These neurons innervate the ipsilateral MB calyx, as well as the lateral horn, lobula, optical track and esophagus, and medial and posterior protocerebrum. They have been implicated in the regulation of courtship behaviors but have no known role in learning and memory. How the multiple dopaminergic circuits that converge on the MB regulate learning is a major question (Boto, 2019).

    This study has investigated the distinct roles of MB-innervating dopaminergic circuits in neuronal plasticity and behavioral memory. PPL2 neurons were found to play a role in learning, modulating neuronal gain and memory strength without imparting a strong valence. Thus, different subsets of dopaminergic neurons converging on memory-encoding neurons (the MB neurons) play roles in modulation of memory strength and valence (Boto, 2019).

    This study provides insight into how PPL2 dopaminergic neurons regulate neuronal plasticity in the MB and behavioral learning. PPL2 neurons project to the MB calyx, where intrinsic MB neurons receive input from olfactory projection neurons. This places them in a position to exert strong influence over MB olfactory responses. The present data suggest that they both act as a gain control that modulates the MB olfactory responses and increase the strength of aversive short-term memory. Activation of PPL2 neurons in a differential conditioning protocol increased the relative responsivity to the paired odor in γ neurons. Behaviorally, this did not drive memory on its own but increased the strength of memory if paired with odor-shock conditioning. Therefore, PPL2 neurons appear to modulate the strength of aversive memory, rather than dictating its content. One mechanism underlying this effect could be that PPL2 neurons enhance MB responses to the odor during training, facilitating the generation of synaptic plasticity that has been observed at the MB output synapses. The memory enhancement effect that this study observed in flies may reflect a more general role that dopaminergic circuits play in other species. For instance, in mice, dopaminergic projections to the medial prefrontal cortex are not sufficient to induce memory, but they improve learning via effects on stimulus discrimination (Popescu et al., 2016) (Boto, 2019).

    Previous studies have suggested that PPL2 neurons could regulate motivation and arousal. Increased responses in the MB do not likely represent the valence of a memory directly, but they may reflect a salience or motivational component of memory. This is supported by PPL1 stimulation failing to induce changes in the odor representation in the MB but inducing conditioned aversion that drives heterosynaptic depression at certain MB-mushroom body output neuron (MBON) synapses. In contrast, PPL2 neurons drive strong Ca2+ response plasticity in the MB but do not encode strong valence on their own. The effect was limited to aversive memory, possibly because the starvation necessary for appetitive protocols had already maximized the animals' arousal state and/or salience of the sensory cues (though other possibilities are discussed later) (Boto, 2019).

    One function of PPL2 dopaminergic neurons may be to regulate the net responsivity of MB γ neurons to odorants and thereby alter the potential for stimuli to drive memory strength. Alternatively, the plasticity could regulate the balance of excitation across downstream MBONs that innervate spatially discrete zones of the MB and drive approach or avoidance behavior. For instance, increasing responses of MB γ neurons alone could increase the net excitatory drive to aversive MBONs relative to appetitive MBONs. In a previous study, appetitive conditioning was found to robustly increase Ca2+ responses to CS+ across the MB lobes (including both γ and α/β) (Louis, 2018). This could be interpreted to indicate either that the motivational component of appetitive conditioning differentially engages MB circuitry relative to aversive conditioning or that the appetitive valence is encoded as a bona fide cellular-level memory trace, comprising an increase in Ca2+ responses across all MB lobes. If the latter is true, perhaps a selective increase in Ca2+ responses in γ reflects a more aversive signature. Previous studies have demonstrated a critical role of γ neurons in short-term memory. Rescue of Rutabaga in the γ lobe of rut mutants is sufficient to restore performance in short-term memory, and rescue of the D1-like DopR receptor in the γ lobe is sufficient to rescue both short- and long-term memory. In addition, aversive learning induces plasticity in synaptic vesicle release from the MB γ lobes (Boto, 2019).

    Several caveats in experimental interpretations should be noted. First, it is not known whether the MB plasticity forms in parallel to memory enhancement or directly drives it. Contributions of polysynaptic circuit elements to the physiological effects (MB plasticity) and/or behavioral effects (enhanced memory) are possible. Future mapping studies may identify additional circuit elements contributing to the memory networks underlying these phenomena. Nonetheless, anatomical innervation of the MB calyx by PPL2 neurons positions them to provide strong modulatory input to the MB dendrites and associated neuronal circuitry. Thus, while valence is layered at the MB output synapses, the data suggest that PPL2 neurons may be a control mechanism that influences how responsive the MB is to odors, potentially altering the propensity for synaptic plasticity downstream (Boto, 2019).

    Reward signaling in a recurrent circuit of dopaminergic neurons and peptidergic Kenyon cells

    Dopaminergic neurons in the brain of the Drosophila larva play a key role in mediating reward information to the mushroom bodies during appetitive olfactory learning and memory. Using optogenetic activation of Kenyon cells, evidence is provided that recurrent signaling exists between Kenyon cells and dopaminergic neurons of the primary protocerebral anterior (pPAM) cluster. Optogenetic activation of Kenyon cells paired with odor stimulation is sufficient to induce appetitive memory. Simultaneous impairment of the dopaminergic pPAM neurons abolishes appetitive memory expression. Thus, it is argued that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. This study further shows that this feedback signaling is dependent on short neuropeptide F, but not on acetylcholine known to be important for odor-shock memories in adult flies. These data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization (Lyutova, 2019).

    Aso, Y., Ray, R. P., Long, X., Bushey, D., Cichewicz, K., Ngo, T. T., Sharp, B., Christoforou, C., Hu, A., Lemire, A. L., Tillberg, P., Hirsh, J., Litwin-Kumar, A. and Rubin, G. M. (2019). Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics. Elife 8. PubMed ID: 31724947

    Nitric oxide acts as a cotransmitter in a subset of dopaminergic neurons to diversify memory dynamics

    Animals employ diverse learning rules and synaptic plasticity dynamics to record temporal and statistical information about the world. However, the molecular mechanisms underlying this diversity are poorly understood. The anatomically defined compartments of the insect mushroom body function as parallel units of associative learning, with different learning rates, memory decay dynamics and flexibility. This study shows that nitric oxide (NO) acts as a neurotransmitter in a subset of dopaminergic neurons in Drosophila. NO's effects develop more slowly than those of dopamine and depend on soluble guanylate cyclase in postsynaptic Kenyon cells. NO acts antagonistically to dopamine; it shortens memory retention and facilitates the rapid updating of memories. The interplay of NO and dopamine enables memories stored in local domains along Kenyon cell axons to be specialized for predicting the value of odors based only on recent events. These results provide key mechanistic insights into how diverse memory dynamics are established in parallel memory systems (Aso, 2019).

    An animal's survival in a dynamically changing world depends on storing distinct sensory information about their environment as well as the temporal and probabilistic relationship between those cues and punishment or reward. Thus it is not surprising that multiple distributed neuronal circuits in the mammalian brain have been shown to process and store distinct facets of information acquired during learning. Even a simple form of associative learning such as fear conditioning induces enduring changes, referred to as memory engrams, in circuits distributed across different brain areas. Do these multiple engrams serve different mnemonic functions, what molecular and circuit mechanisms underlie these differences, and how are they integrated to control behavior? Localizing these distributed engrams, understanding what information is stored in each individual memory unit and how units interact to function as one network are important but highly challenging problems. The Drosophila mushroom body (MB) provides a well-characterized and experimentally tractable system to study parallel memory circuits. Olfactory memory formation and retrieval in insects requires the MB. In associative olfactory learning, exposure to an odor paired with a reward or punishment results in formation of a positive- or negative-valence memory, respectively. In the MB, sensory stimuli are represented by the sparse activity of ~2,000 Kenyon cells (KCs). Each of 20 types of dopaminergic neurons (DANs) innervates compartmental regions along the parallel axonal fibers of the KCs. Similarly, types of mushroom body output neurons (MBONs) arborize their dendrites in specific axonal segments of the KCs; together, the arbors of the DANs and MBONs define the compartmental units of the MB. Activation of individual MBONs can cause behavioral attraction or repulsion, depending on the compartment in which their dendrites arborize, and MBONs appear to use a population code to govern behavior (Aso, 2019).

    A large body of evidence indicates that these anatomically defined compartments of the MB are also the units of associative learning. Despite the long history of behavioral genetics in fly learning and memory, many aspects of the signaling pathways governing plasticity -- especially whether they differ between compartments -- remain poorly understood. Nevertheless, dopaminergic neurons and signaling play a key role in all MB compartments, and flies can be trained to form associative memories by pairing the presentation of an odor with stimulation of a single dopaminergic neuron. Punishment or reward activates distinct sets of DANs that innervate specific compartments of the MB. Activation of the DAN innervating a MB compartment induces enduring depression of KC-MBONs synapses in those specific KCs that were active in that compartment at the time of dopamine release. Thus, which compartment receives dopamine during training appears to determine the valence of the memory, while which KCs were active during training determines the sensory specificity of the memory (Aso, 2019).

    Compartments operate with distinct learning rules. Selective activation of DANs innervating specific compartments has revealed that they can differ extensively in their rates of memory formation, decay dynamics, storage capacity, and flexibility to learn new associations. For instance, the dopaminergic neuron PAM-α1 can induce a 24h memory with a single 1-minute training session, whereas PPL1-α3 requires ten repetitions of the same training to induce a 24h memory. PPL1-γ1pedc (aka MB-MP1) can induce a robust short-lasting memory with a single 10-second training, but cannot induce long-term memories even after 10 repetitions of a 1-minute training. PAM-α1 can write a new memory without compromising an existing memory, whereas PPL1-γ1pedc extinguishes the existing memory when writing a new memory. What molecularand cellular differences are responsible for the functional diversity of these compartments? Some differences might arise from differences among KC cell types, but memory dynamics are different even between compartments that lie along the axon bundles of the same Kenyon cells (for example, α1 and α3). This paper shows that differences in memory dynamics between MB compartments can arise from the deployment of distinct cotransmitters by the DAN cell types that innervate them (Aso, 2019).

    Evidence from a wide range of organisms establishes that dopaminergic neurons often release a second neurotransmitter, but the role of such cotransmitters in diversifying neuronal signaling is much less clear. In rodents, subsets of dopaminergic neurons co-release glutamate or GABA. In mice and Drosophila, single-cell expression profiling reveals expression of diverse neuropeptides in dopaminergic neurons. EM connectome studies of the mushroom body in adult and larval Drosophila reveal the co-existence of small-clear-core and large- dense-core synaptic vesicles in individual terminals of dopaminergic neurons; moreover, the size of the observed large-dense-core 02 vesicles differs between DAN cell types (Aso, 2019).

    This study found that NOS, the enzyme that synthesizes NO, was located in the terminals of a subset of DAN cell types. NOS catalyzes the production of nitric oxide (NO) from L-arginine. Drosophila NOS is regulated by Ca2+/calmodulin, raising the possibility that NO synthesis might be activity dependent. Furthermore, the localization of the NOS1 protein in the axonal terminals of DANs is consistent with NO serving as a cotransmitter. The conclusion that NO acts as a neurotransmitter is supported by the observation that NO signaling requires the presence of a putative receptor, soluble guanylate cyclase, in the postsynaptic Kenyon cells. This role contrasts with the proposed cell-autonomous action of NOS in the ellipsoid body, in which NO appears to target proteins within the NOS-expressing ring neurons themselves, rather than conveying a signal to neighboring cells. The valence-inversion phenotype observed when PPL1-γ1pedc was optogenetically activated in a dopamine-deficient background can be most easily explained if NO induces synaptic potentiation between odor-activated KCs and their target MBONs. Modeling work is consistent with this idea, but testing this idea and 19 other possible mechanisms for NO action will require physiological experiments (Aso, 2019).

    During olfactory learning, the concentration of Ca2+ in KC axons represents olfactory information. The coincidence of a Ca2+ rise in spiking KCs and activation of the G-protein-coupled Dop1R1 dopamine receptor increases adenylyl cyclase activity. The resultant cAMP in turn activates protein kinase A, a signaling cascade that is important for synaptic plasticity and memory formation throughout the animal phyla. In contrast, when DANs are activated without KC activity, and thus during low intracellular Ca2+ in the KCs, molecular pathways involving the Dop1R2 receptor, Rac1 and Scribble facilitate decay of memory (Aso, 2019).

    This study found that NOS in PPL1-γ1pedc shortens memory retention, while facilitating fast updating of memories in response to new experiences. These observations could be interpreted as indicating that NO regulates forgetting. Indeed, NO-dependent effect requires scribble in KCs, a gene previously reported as a component of active forgetting. However, it is an open question whether the signaling pathways for forgetting, which presumably induce recovery from synaptic depression, are related to signaling cascades downstream of NO and guanylate cyclase, which appear to be able to induce memory without prior induction ofsynaptic depression by dopamine. Lack of detectable 1-day memory formation after spaced training with PPL1-γ1pedc can be viewed as a balance between two distinct, parallel biochemical signals, one induced by dopamine and the other by NO, rather than the loss of information (that is, forgetting). Confirming this interpretation will require better understanding of the signaling pathways downstream of dopamine and NO. The search for such pathways will be informed by the prediction from modeling that dopamine and NO may alter two independent parameters that define synaptic weights with a multiplicative interaction (Aso, 2019).

    In the vertebrate cerebellum, which has many architectural similarities to the MB, long-term-depression at parallel fiber-Purkinje cell synapses (equivalent to KC-MBON synapses) induced by climbing fibers (equivalent to DANs) can coexist with long-term-potentiation by NO. In this case, the unaltered net synaptic weight results from a balance between coexisting LTD and LTP rather than recovery from LTD. This balance was suggested to play an important role in preventing memory saturation in the cerebellum 59 and allowing reversal of motor learning. In the Drosophila MB, a similar facilitation was observed of reversal learning by NO. The antagonistic roles of NO and synaptic depression may be a yet another common feature of the mushroom body and the cerebellum (Aso, 2019).

    Opposing cotransmitters have been observed widely in both invertebrate and vertebrate neurons. A common feature in these cases is that the transmitters have distinct time courses of action. For instance, hypothalamic hypocretin-dynorphin neurons that are critical for sleep and arousal synthesize excitatory hypocretin and inhibitory dynorphin. When they are released together repeatedly, the distinct kinetics of their receptors result in an initial outward current, then little current, and then an inward current in the postsynaptic cells. In line with these observations, this study found that dopamine and NO show distinct temporal dynamics: NO-dependent memory requires repetitive training and takes longer to develop than dopamine-dependent memory. What molecular mechanisms underlie these differences? Activation of NOS may require stronger or more prolonged DAN activation than does dopamine release. Alternatively, efficient induction of the signaling cascade in the postsynaptic KCs might require repetitive waves of NO input. Direct measurements of release of dopamine and NO, and downstream signaling events by novel sensors will be needed to address these open questions (Aso, 2019).

    Decades of behavioral genetic studies have identified more than one hundred genes underlying olfactory conditioning in Drosophila. Mutant andtargeted rescue studies have been used to map the function of many memory-related genes encoding synaptic or intracellular signaling proteins (for example, rutabaga, DopR1/dumb, DopR2/DAMB, PKA-C1/DC0, Synapsin, Bruchpilot, Orb2 and Rac1) to specific subsets of Kenyon cells. However, it is largely unknown if these proteins physically colocalize at the same KC synapses to form intracellular signaling cascades. Some of 97 these proteins might preferentially localize to specific MB compartments. Alternatively, they may distribute uniformly along the axon of Kenyon cells, but be activated in only specific compartments. Identification of cell-type-specific cotransmitters in DANs enabled a beginning to the exploration of this question (Aso, 2019).

    Optogenetic activation of specific DANs was used to induce memory in specific MB compartments, while manipulating genes in specific types of KCs. This approach allowed mapping and characterization of the function of memory-related genes at a subcellular level. For example, the Gycbeta100B gene, which encodes a subunit of sGC, has been identified as 'memory suppressor gene' that enhances memory retention when pan-neuronally knocked down, but the site of its action was unknown. Gycbeta100B appears to be broadly dispersed throughout KC axons, based on the observed distribution of a Gycbeta100B-EGFP fusion protein. The experiments ectopically expressing NOS in PPL1-α3 DANs that do not normally signal with NO is most easily explained if sGC is available for activation in all MB compartments. What are the molecular pathways downstream to cGMP? How do dopamine and NO signaling pathways interact in regulation of KC synapses? Previous studies and RNA-Seq data suggest several points of possible crosstalk. In cultured KCs from cricket brains, cGMP-dependent protein kinase (PKG) mediates NO-induced augmentation of a Ca2+ channel current. However, no expression of either of the genes encoding Drosophila PKGs (foraging and Pkg21D) was detected in KCs in RNA-Seq studies. On the other hand, cyclic nucleotide-gated channels and the cGMP-specific phosphodiesterase Pde9 are expressed in KCs. Biochemical studies have shown that the activity of sGC is calcium dependent and that PKA can enhance the NO-induced activity of sGC by phosphorylating sGC; sGC isolated from flies mutant for adenylate cyclase, rutabaga, show lower activity than sGC from wild-type brains, suggesting crosstalk between the cAMP and cGMP pathways (Aso, 2019).

    All memory systems must contend with a tension between the strength and longevity of the memories they form. The formation of a strong immediate memory interferes with and shortens the lifetimes of previously formed memories, and reducing this interference requires a reduction in initial memory strength that can only be overcome through repeated exposure. Theoretical studies have argued that this tension can be resolved by memory systems that exhibit a heterogeneity of timescales, balancing the need for both fast, labile memory and slow consolidation of reliable memories. The mechanisms responsible for this heterogeneity, and whether they arise from complex signaling within synapses themselves), heterogeneity across brain areas, or both, have not been identified (Aso, 2019).

    NO acts antagonistically to dopamine and reduces memory retention while facilitating the rapid updating of memory following a new experience. Viewed in isolation, the NO-dependent reduction in memory retention within a single compartment may seem disadvantageous, but in the presence of parallel learning pathways, this shortened retention may represent a key mechanism for the generation of multiple memory timescales that are crucial for effective learning. During shock conditioning, for example, multiple DANs respond to the aversive stimulus, including PPL1-γ1pedc, PPL1-γ2α'1, PPL1-α3. This study has shown that optogenetic activation of these DAN cell types individually induces negative-valence olfactory memory with distinct learning rates. The NOS-expressing PPL1-γ1pedc induces memory with the fastest learning rate in a wild-type background, and this study shows that it induces an NO-dependent memory trace when dopamine synthesis is blocked, with a much slower learning rate and opposite valence (Aso, 2019).

    Robust and stable NO-dependent effects were only observed when training was repeated 10 times. Under such repeated training, compartments with slower learning rates, such as α3, form memory traces in parallel to those formed in γ1pedc. Thus, flies may benefit from the fast and labile memory formed in γ1pedc without suffering the potential disadvantages of 58 shortened memory retention, as long-term memories are formed in parallel in other compartments. The Drosophila MB provides a tractable experimental system to study the mechanisms and benefits of diversifying learning rate, retention, and flexibility in parallel memory units, as well as exploring how the outputs from such unitsare integrated to drive behavior (Aso, 2019).

    A neural mechanism for deprivation state-specific expression of relevant memories in Drosophila

    Motivational states modulate how animals value sensory stimuli and engage in goal-directed behaviors. The motivational states of thirst and hunger are represented in the brain by shared and unique neuromodulatory systems. However, it is unclear how such systems interact to coordinate the expression of appropriate state-specific behavior. The activity of two brain neurons expressing leucokinin neuropeptide is elevated in thirsty and hungry flies, and leucokinin release is necessary for state-dependent expression of water- and sugar-seeking memories. Leucokinin inhibits two types of mushroom-body-innervating dopaminergic neurons (DANs) to promote thirst-specific water memory expression, whereas it activates other mushroom-body-innervating DANs to facilitate hunger-dependent sugar memory expression. Selection of hunger- or thirst-appropriate memory emerges from competition between leucokinin and other neuromodulatory hunger signals at the level of the DANs. Therefore, coordinated modulation of the dopaminergic system allows flies to prioritize the expression of the relevant state-dependent motivated behavior (Senapati, 2019).

    Dopaminergic neurons can influence heat-box place learning in Drosophila

    Dopamine provides crucial neuromodulatory functions in several insect and rodent learning and memory paradigms. However, an early study suggested that dopamine may be dispensable for aversive place memory in Drosophila. This study tested the involvement of particular dopaminergic neurons in place learning and memory. The thermogenetic tool Gr28bD was used to activate protocerebral anterior medial (PAM) cluster and non-PAM dopaminergic neurons in an operant way in heat-box place learning. Activation of PAM neurons influences performance during place learning, but not during memory testing. These findings provide a gateway to explore how dopamine influences place learning (Mishra, 2020).

    Circuits that encode and guide alcohol-associated preference

    A powerful feature of adaptive memory is its inherent flexibility. Alcohol and other addictive substances can remold neural circuits important for memory to reduce this flexibility. However, the mechanism through which pertinent circuits are selected and shaped remains unclear. This study shows that circuits required for alcohol-associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice in Drosophila melanogaster. During memory expression, subsets of dopamine neurons directly and indirectly modulate the activity of interconnected glutamatergic and cholinergic mushroom body output neurons (MBON). Transsynaptic tracing of neurons important for memory expression revealed a convergent center of memory consolidation within the mushroom body (MB) implicated in arousal, and a structure outside the MB implicated in integration of naive and learned responses. These findings provide a circuit framework through which dopamine neuronal activation shifts from reward delivery to cue onset, and provide insight into the maladaptive nature of memory (Scaplen, 2020).

    A feedforward circuit regulates action selection of pre-mating courtship behavior in female Drosophila

    In the early phase of courtship, female fruit flies exhibit an acute rejection response to avoid unfavorable mating. This pre-mating rejection response is evolutionarily paralleled across species, but the molecular and neuronal basis of that behavior is unclear. This study shows that a putative incoherent feedforward circuit comprising ellipsoid body neurons, cholinergic R4d, and its repressor GABAergic R2/R4m neurons regulates the pre-mating rejection response in the virgin female Drosophila melanogaster. Both R4d and R2/R4m are positively regulated, via specific dopamine receptors, by a subset of neurons in the dopaminergic PPM3 cluster. Genetic deprivation of GABAergic signal via GABAA receptor RNA interference in this circuit induces a massive rejection response, whereas activation of GABAergic R2/R4m or suppression of cholinergic R4d increases receptivity. Moreover, glutamatergic signaling via N-methyl-d-aspartate receptors induces NO-mediated retrograde regulation potentially from R4d to R2/R4m, likely providing flexible control of the behavioral switching from rejection to acceptance. This study elucidates the molecular and neural mechanisms regulating the behavioral selection process of the pre-mating female (Ishimoto, 2020).

    Overall, the present findings provide evidence for a neural relation that regulates the action selection of pre-mating behaviors in female Drosophila. The PPM3, a subset of DA neurons, forms a circuit with the R neurons R2/R4m and R4d in the EB. These different types of R neurons require different types of DA receptors, Dop1R1/D2R and Dop1R2. The knockdown of each DA receptor type indicates that all of these receptors are required to activate the expressing neurons, although the D2R conventionally inhibits D2R-expressing neurons. R2/R4m and R4d are GABAergic and cholinergic, respectively. Synaptic GRASP analysis revealed a neuronal connection from R2/R4m to R4d. R4d inhibition via the GABAA receptor is required for the proper reduction of pre-mating rejection. In addition to DA regulation of R2/R4m, the potential retrograde regulation may facilitate the GABA transmission of R2/R4m, depending on the R4d activity, with the production of NO via NMDAR/NOS signaling. This potentiation-like regulation provides the activation order of each R neuron with flexibility for the neural circuit output, and therefore to the rejection response for controlling the pre-mating behavioral kinetics (Ishimoto, 2020).

    The pre-mating rejection should continue if the encounter does not match the female's criteria. Pheromones are important sexual cues provided by the male. Fruit flies produce cuticular hydrocarbons as pheromonal substances. cVA is a male-specific pheromonal cue that elicits female sexual arousal via the olfactory sensory system. This cVA signal activates pCd via a third-order olfactory interneuron, aSP-g. The SMP region contains aSP-g, pCd, and PPM3, although the direct connection between them has not been demonstrated. This aggregation of important components for female mating behavior suggests that pCd potentially integrates and verifies male information to execute the final decision for initiating the copulation. The female must resist the encounter until the evaluation process is complete-that is, the pre-mating rejection response controlled by the circuit found in the present study. These considerations lead to an assumption that the pCd and PPM3/R neuron circuits execute pre-mating computation in parallel. Many sexually dimorphic behaviors are reportedly mediated by sex-specific neural circuits (e.g., pCd, pC1). PPM3 and R neurons do not express fruitless or dsx, and no morphological sexual dimorphism has been detected. Thus, a non-sexually dimorphic circuit modulating sex-specific pre-mating behavior is proposed(Ishimoto, 2020).

    Genetic manipulations of R neurons altered the pre-mating kinetics in virgin female flies, leading to the question of whether the modulation of R neurons also affects post-mating behavior, which is induced by injection of the seminal fluid sex peptide from the male fly and sustained for several days. One of the remarkable characteristics of post-mating rejection is ovipositor extrusion controlled by a distinct class of neurons in post-mating females, but either suppression of R2/R4m neurons or activation of R4d neurons, both of which reduced receptivity, rarely induced ovipositor extrusion. Mated females with R4d suppression, which makes virgin females highly receptive, exhibited a large rejection response similar to that in mated control females, and no copulation was observed during the 1-h observation time. In addition to these observations, the virgin females with Rdl knockdown in R4d reduced their walking speed with the progression of the male courtship; however, the walking speed of the mated females increases with the progression of the male courtship. Although further studies are required to elucidate the whole picture of rejection control in females, neuronal regulation of the post-mating response is presumably parallel to the pre-mating rejection response modulated by R neurons in the EB (Ishimoto, 2020).

    This study found circuit functions that contain PPM3 DA subcluster neurons, and R neurons R2/R4m and R4d, were involved in the regulation of the kinetics of pre-mating behavior in virgin females. Pre-mating rejection is acutely elicited and then gradually decreased. A circuital feature of PPM3/R neurons may provide a theoretical action for controlling these pre-mating kinetics. PPM3 sends DA signals to both R2/R4m and R4d, and this recurrent circuit forms a feedforward motif with a repressor, the so-called incoherent type 1 feedforward loop (I1-FFL). I1-FFL is one of the most common network motifs implicated in gene and protein regulation networks, metabolic pathways, and neural networks from bacteria to humans. In the I1-FFL circuit, an activator X (e.g., PPM3) activates a target Z (e.g., R4d) and simultaneously activates another target Y (e.g., R2/R4m) that inhibits the target Z. Previous studies demonstrated that the I1-FFL accelerates the response of target Z, with a shorter response time. Moreover, upon input from X, Z activity increases and then, depending on the Y activity, it decreases toward basal levels. The I1-FFL circuit comprising PPM3, R2/R4m, and R4d would thus theoretically generate the acute activation of R4d, which promotes the pre-mating rejection behavior of females that would later be gradually attenuated by GABAergic signals from R2/R4m. This schema is consistent with the current findings, in which the circuit of PPM3/R neurons plays an important role in the action selection of pre-mating behavior (Ishimoto, 2020).

    Genetic analysis indicates that retrograde signals from R4d to R2/R4m, mediated by NO and NMDAR, would add some flexibility to the Y-to-Z regulation in the I1-FFL circuit, contributing to progressive attenuation of the rejection response in virgin females. Depolarization of R4d activates NMDAR by the coincident input of glutamate from R2/R4m. The retrograde signals induced by NMDAR/NOS/sGC probably facilitate the GABA transmission of R2/R4m, which progressively suppresses R4d activity via the GABAA receptors. Because NMDARs require simultaneous activation by glutamate and depolarization, the NMDAR pore opening may be insufficient until the activity of R4d neurons will reach a sufficient level. This may lead to keeping the GABAergic R2/R4m function at a lower level that is under a certain threshold required for GABA release, and it may induce a continuous pre-mating rejection response. Recently, new subdivision of R neurons (R5 and R6) were classified by clonal morphological analysis and cell lineage analysis. These new subclasses of R neurons may provide a novel function of the putative I1-FFL. To investigate this further, the physiological properties of each R neuron should be analyzed (Ishimoto, 2020).

    The distribution pattern of neurotransmitters in EB neurons has been documented. In the R2/R4m axonal ring structure, the glutamatergic population seems to be smaller than the GABAergic population, suggesting that the neuronal population in the R2/R4m is heterologous and contains few, if any, neurons that co-express both glutamate and GABA. In the vertebrate system, heterosynaptic regulation of GABAergic transmission is incorporated into inhibitory long-term potentiation. Inhibitory long-term potentiation of GABAergic neurons is induced at heterosynaptic sites containing glutamatergic and GABAergic neurons as presynaptic cells. In GABAergic inhibitory long-term potentiation, NO induced by glutamatergic activation of the NMDAR/NOS pathway retrogradely activates sGC, which augments cGMP levels to enhance GABA release. These intriguing analogies between vertebrate findings and the current results suggest that a similar molecular machinery potentiates GABAergic subsets of the R2/R4m neurons for attenuating the pre-mating rejection response, and hence for the action selection of pre-mating kinetics. Regarding the action selection for adaptive behaviors, others have proposed a correspondence of functions and neural architectures between vertebrate basal ganglia and the insect central complex, which contains EB neurons. In the basal ganglia, DA activates the neurons of the nucleus accumbens with modulation of glutamate and GABA release. The neurochemicals in the nucleus accumbens released by sexual interaction regulate female action selection for the affiliation of monogamous prairie voles (Microtus ochrogaster). The current genetic manipulations of particular DA, cholinergic, glutamatergic, and GABAergic systems in the central complex significantly affected the action selection for female fruit fly pre-mating behaviors, implying that the mechanisms are similar to those in the vertebrate system, which sheds light on the evolutionary parallels and diversities across the animal kingdom (Ishimoto, 2020).

    Distinct dopamine receptor pathways underlie the temporal sensitivity of associative learning

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

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

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

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

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

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

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

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

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

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

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

    Input Connectivity Reveals Additional Heterogeneity of Dopaminergic Reinforcement in Drosophila

    Different types of Drosophila dopaminergic neurons (DANs) reinforce memories of unique valence and provide state-dependent motivational control. Prior studies suggest that the compartment architecture of the mushroom body (MB) is the relevant resolution for distinct DAN functions. This study used a recent electron microscope volume of the fly brain to reconstruct the fine anatomy of individual DANs within three MB compartments. The 20 DANs of the γ5 compartment, at least some of which provide reward teaching signals, can be clustered into 5 anatomical subtypes that innervate different regions within γ5. Reconstructing 821 upstream neurons reveals input selectivity, supporting the functional relevance of DAN sub-classification. Only one PAM-γ5 DAN subtype γ5(fb) receives direct recurrent feedback from γ5β'2a mushroom body output neurons (MBONs) and behavioral experiments distinguish a role for these DANs in memory revaluation from those reinforcing sugar memory. Other DAN subtypes receive major, and potentially reinforcing, inputs from putative gustatory interneurons or lateral horn neurons, which can also relay indirect feedback from MBONs. The single aversively reinforcing PPL1-γ1pedc DAN was similarly reconstructed. The γ1pedc DAN inputs mostly differ from those of γ5 DANs and they cluster onto distinct dendritic branches, presumably separating its established roles in aversive reinforcement and appetitive motivation. Tracing also identified neurons that provide broad input to γ5, β'2a, and γ1pedc DANs, suggesting that distributed DAN populations can be coordinately regulated. These connectomic and behavioral analyses therefore reveal further complexity of dopaminergic reinforcement circuits between and within MB compartments (Otto, 2020).

    Carbon monoxide, a retrograde messenger generated in postsynaptic mushroom body neurons, evokes noncanonical dopamine release

    Dopaminergic neurons innervate extensive areas of the brain and release dopamine (DA) onto a wide range of target neurons. However, DA release is also precisely regulated. In Drosophila melanogaster brain explant preparations, DA is released specifically onto α3/α'3 compartments of mushroom body (MB) neurons that have been coincidentally activated by cholinergic and glutamatergic inputs. The mechanism for this precise release has been unclear. This study found that coincidentally activated MB neurons generate carbon monoxide (CO), which functions as a retrograde signal evoking local DA release from presynaptic terminals. CO production depends on activity of heme oxygenase in postsynaptic MB neurons, and CO-evoked DA release requires Ca(2+) efflux through ryanodine receptors in DA terminals. CO is only produced in MB areas receiving coincident activation, and removal of CO using scavengers blocks DA release. It is proposed that DA neurons use two distinct modes of transmission to produce global and local DA signaling (Ueno, 2020).

    Dopamine (DA) is required for various brain functions, including the regulation of global brain states, such as arousal and moods. To perform these functions, individual DA neurons innervate extensive areas of the brain, and release DA onto a wide range of target neurons through a processes known as volume transmission. However, this extensive innervation is not suitable for precise, localized release of DA, and it has been unclear how widely innervating dopaminergic neurons can also direct DA release onto specific target neurons (Ueno, 2020).

    In Drosophila, olfactory associative memories are formed and stored in the mushroom bodies (MBs) where Kenyon cells, MB intrinsic neurons which are activated by different odors, form synaptic connections with various MB output neurons, which regulate approach and avoidance behaviors. Dopaminergic neurons (DA neurons) modulate plasticity of synapses between Kenyon cells and MB output neurons. However, while there are ~2000-2500 Kenyon cells that form thousands of synapses with MB output neurons, plasticity at these synapses is regulated by relatively few DA neurons. This indicates that canonical action potential-dependent release cannot fully explain DA release and plasticity. It was recently determined that in Drosophila, synaptic vesicular (SV) exocytosis from DA terminals is restricted to MB neurons that have been activated by coincident inputs from odor-activated cholinergic pathways, and glutamatergic pathways, which convey somatosensory (pain) information. Odor information is transmitted to the MBs by projection neurons from the antennal lobe (AL), while somatosensory information is transmitted to the brain via ascending fibers of the ventral nerve cord (AFV). AL stimulation evokes Ca2+ responses in the MB by activating nAChRs, and AFV stimulation evokes Ca2+ responses in the MBs by activating N-Methyl-D-aspartate receptors (NMDARs) in the MBs. Significantly, when the AL and AFV are stimulated simultaneously (AL 1 AFV) or the AL and NMDARs are stimulated simultaneously (AL 1 NMDA), plasticity occurs such that subsequent AL stimulations causes increased Ca2+ responses in the a3/a93 compartments. This plasticity is known as long-term enhancement (LTE) of MB responses and requires activation of D1 type DA receptors (D1Rs) in the MBs. Furthermore, while activation of D1Rs alone is sufficient to produce LTE, neither AL nor AFV stimulation alone is able to cause SV exocytosis from presynaptic DA terminals projecting onto the a3/a93 compartments of the vertical MB lobes. Instead, exocytosis from DA terminals occurs only when postsynaptic Kenyon cells are activated by coincident AL 1 AFV or AL 1 NMDA stimulation. Strikingly, while MBs are bilateral structures and DA neurons project terminals onto both sides of MBs, SV exocytosis occurs specifically in MB areas that have been coincidently activated. Based on these results, it is proposed that coincident inputs specify the location where DA is released, whereas DA induces plastic changes needed to encode associations. However, it has been unclear how activated Kenyon cells send a retrograde 'demand' signal to presynaptic DA terminals to induce SV release (Ueno, 2020).

    This study used a Drosophila dissected brain system to examine synaptic plasticity and DA release, and found that coincidentally activated postsynaptic Kenyon cells generate the retrograde messenger, carbon monoxide (CO). CO is generated by heme oxygenase (HO) in postsynaptic MB neurons, and induces DA release from presynaptic terminals by evoking Ca2+ release from internal stores via ryanodine receptors (RyRs). Thus, while individual DA neurons extensively innervate the MBs, on-demand SV exocytosis allows DA neurons to induce plasticity in specific target neurons (Ueno, 2020).

    CO functions as a retrograde on-demand messenger for SV exocytosis in presynaptic DA terminals A central tenet of neurobiology is that action potentials, propagating from the cell bodies, induce Ca2+ influx in presynaptic terminals to evoke SV exocytosis. However, recent mammalian studies have shown that only a certain fraction of a large number of presynaptic DA release sites is involved in canonical SV exocytosis. In this study, a novel mechanism of SV exocytosis was identified in which activity in postsynaptic neurons evokes presynaptic release to induce plastic changes. This mechanism allows the timing and location of DA release to be strictly defined by activity of postsynaptic neurons (Ueno, 2020).

    On-demand SV exocytosis uses CO as a retrograde signal from postsynaptic MB neurons to presynaptic DA terminals. CO fulfills the criteria that have been proposed for a retrograde messenger: (1) it was demonstrated that HO, which catalyzes CO production, is highly expressed in postsynaptic MB neurons, indicating that MB neurons have the capacity to synthesize the messenger; (2) it was shown that pharmacological and genetic suppression of HO activity in the MBs inhibits CO production, presynaptic DA release, and LTE, and (3) using a CO fluorescent probe, COP-1, it was demonstrated that CO is generated in the MBs following coincident stimulation of the MBs, and CO generation is restricted to lobes of MB neurons that receive coincident stimulation. It was further shown that direct application of CO, or a CO donor, induces DA release from presynaptic terminals, whereas addition of a CO scavenger, HemoCD, suppresses release. Fourth, it was demonstrated that CO activates RyR in presynaptic terminals to induce SV exocytosis. Strikingly, CO-dependent SV exocytosis does not depend on influx of extracellular Ca2+ but instead requires efflux of Ca2+ from internal stores via RyR. Finally, it was shown that pharmacological inhibition and genetic suppression of RyR in DA neurons impair DA release after coincident stimulation and CO application (Ueno, 2020).

    Other retrograde signals, such as NO and endocannabinoids, enhance or suppress canonical SV exocytosis, but this study finds that CO-dependent DA release occurs even in conditions that block neuronal activity and Ca2+ influx in presynaptic DA terminals. This suggests that CO does not function to modulate canonical SV exocytosis, but may instead evoke exocytosis through a novel mechanism. Several previous studies have indicated that CO and RyR-dependent DA release also occurs in mammals. A microdialysis study has shown that CO increases the extracellular DA concentration in the rat striatum and hippocampus, either through increased DA release or inhibition of DA reuptake. Also, pharmacological stimulation of RyRs has been reported to induce DA release in the mice striatum. This release is attenuated in RyR3-deficient mice, while KCl-induced DA release, which requires influx of extracellular Ca2+, is unaffected, suggesting that RyR-dependent release is distinct from canonical DA release. However, it has been unknown whether and how CO is generated endogenously. Physiologic conditions that activate RyR to evoke DA release have also been unclear (Ueno, 2020).

    While most neurotransmitters are stored in synaptic vesicles and released on neuronal depolarization, the release of gaseous retrograde messengers, such as NO and CO, is likely coupled to activation of their biosynthetic enzymes, NOS and HO. In mammals, an HO isoform, HO-2, is selectively enriched in neurons, and HO-2-derived CO is reported to function in plasticity. HO-2 is activated by Ca2+/calmodulin (CaM) binding, and by casein kinase II (CKII) phosphorylation. Previously, it was shown that coincident AL 1 NMDA stimulation induces a robust Ca2+ increase in the MBs that is greater than the increase from either stimulation alone (Ueno, 2017). It is proposed that this increase may activate Drosophila HO in the MB to generate CO during associative stimulation. While Drosophila has a single isoform of RyR, mammals have three isoforms, RyR1-RyR3. Skeletal muscle and cardiac muscle primarily express RyR1 and RyR2, and the brain, including the striatum, hippocampus, and cortex, expresses all three isoforms. RyRs are known to be activated by Ca2+ to mediate Ca2+ induced Ca2+ release. However, CO-evoked DA release occurs even in the presence of Ca2+-free extracellular solutions containing TTX and EGTA, suggesting that CO activates RyR through a different mechanism. In addition to Ca2+, RyR can be activated by calmodulin, ATP, PKA, PKG, cADP-ribose, and NO. NO can directly stimulate RyR1 nonenzymatically by S-nitrosylating a histidine residue to induce Ca2+ efflux. CO has been reported to activate Ca2+-activated potassium channels (KCa) through a nonenzymatic reaction in rat artery smooth muscle, raising the possibility that it may activate RyR through a similar mechanism. Alternatively, both NO and CO can bind to the heme moiety of soluble guanlylate cyclase leading to its activation. Activated soluble guanlylate cyclase produces cGMP, and cGMP-dependent protein kinase (PKG) rapidly phosphorylates and activates RyRs. Interestingly, NO increases DA in the mammalian striatum in a neural activity-independent manner. Since activation of RyRs also increases extracellular DA in the striatum, hippocampus, and cortex, NO may play a pivotal role in RyRs activation and DA release in mammals. However, NOS expression has not been detected in the MBs, suggesting that, in Drosophila, CO rather than NO may function in this process (Ueno, 2020).

    Previous studies have shown that electrical activity from the AL and AFV is transmitted to the MBs by cholinergic and glutamatergic neurons acting on nAChRs and NMDARs, respectively. Although the cholinergic inputs from the AL are known to be delivered by projection neurons, the glutamate inputs are still unclear. Previous work identified glutamatergic neurons that innervate a3/a93 compartments of the MBs and show SV release on electrical stimulation of the AFV (Ueno, 2017). It is proposed that these neurons may transmit information regarding AFV stimuli to the MBs. Alternatively, while NMDARs are localized throughout MB lobes, vesicular glutamate transporter-positive terminals are found only sparsely on the MBs. This suggests that neurons expressing a currently uncharacterized vesicular glutamate transporter may convey information from the AFV to MBs (Ueno, 2020).

    DA plays a critical role in associative learning and synaptic plasticity. In flies, neutral odors induce MB responses by activating sparse subsets of MB neurons. After being paired with electrical shocks during aversive olfactory conditioning, odors induce larger MB responses in certain areas of the MBs. This plastic change was modeled in ex vivo brains as LTE, and it was shown that DA application alone is sufficient to induce this larger response (Ueno, 2017). However, in the Drosophila brain, only a small number of DA neurons (~12 for aversive and ~100 for appetitive) regulate plasticity in ~2000 MB Kenyon cells. Thus, to form odor-specific associations, there should be a mechanism regulating release at individual synapses. CO-dependent on-demand DA release provides this type of control. If on-demand release is involved in plasticity and associative learning, knockdown of genes associated with release should affect learning. Indeed, this study shows that knocking down either dHO in the MBs or RyR in DA neurons impairs olfactory conditioning. While these knockdowns did not completely abolish olfactory conditioning, this may be due to inefficiency of the knockdown lines. Alternatively, on-demand release may not be the only mechanism responsible for memory formation, but may instead be required for a specific phase of olfactory memory (Ueno, 2020).

    In ex vivo studies, this study found that DA release requires coincident activation of postsynaptic MB neurons by cholinergic and glutamatergic stimuli. However, other in vivo imaging studies have shown that DA neurons can be activated and release DA on odor stimulation or shock application alone. Notably, projection of DA terminals is compartmentalized on the MB lobes and shows distinct responses and DA release during sensory processing. In these studies, dopaminergic neurons innervating the the a3/a93 compartments at the tips of the MB vertical lobes were examined, whereas other studies focused on compartments located on the MB horizontal lobes. This suggests that plasticity in different MB compartments may be regulated by different mechanisms. Unfortunately, due the location of the microelectrode for AL stimulation which caused interference in fluorescent imaging of the horizontal lobes, it was not possible to obtain reliable imaging data from these lobes in this study. Another difference between ex vivo and in vivo studies is that in vivo imaging studies use living, tethered, dissected flies that are likely in different states of arousal/distress, are exposed to many different stimuli, and can form unintended associations. In contrast, brains in ex vivo preparations are in a more controlled environment and are likely exposed to fewer unintended sensory stimuli. This may also explain apparent discrepancies between ex vivo and previous in vivo results (Ueno, 2020).

    In mammals, the role of CO in synaptic plasticity is unclear. Application of CO paired with low-frequency stimulation induces LTP, while inhibiting HO blocks LTP in the CA1 region of the hippocampus. However, HO-2-deficient mice have been reported to have normal hippocampal CA1 LTP. In contrast to CO, a role for NO in synaptic plasticity and learning has been previously reported. Thus, at this point, it is an open question whether CO or NO evokes DA release in mammals. Downstream from CO or NO, RyRs have been shown to be required for hippocampal and cerebellar synaptic plasticity (Ueno, 2020).

    The current results suggest that DA neurons release DA via two distinct mechanisms: canonical exocytosis and on-demand release. Canonical exocytosis is evoked by electrical activity of presynaptic DA neurons, requires Ca2+ influx, and may be involved in volume transmission. This mode of release can activate widespread targets over time, and is suited for regulating global brain functions. In contrast, on-demand release is evoked by activity of postsynaptic neurons, requires Ca2+ efflux via RyR, and can regulate function of specific targets at precise times. DA neurons may differentially use these two modes of SV exocytosis in a context-dependent manner. Understanding how DA neurons differentially use these modes of transmission will provide new insights into how a relatively small number of DA neurons can control numerous different brain functions (Ueno, 2020).

    Serotonin receptor 5-HT7 in Drosophila mushroom body neurons mediates larval appetitive olfactory learning

    Serotonin (5-HT) and dopamine are critical neuromodulators known to regulate a range of behaviors in invertebrates and mammals, such as learning and memory. Effects of both serotonin and dopamine are mediated largely through their downstream G-protein coupled receptors through cAMP-PKA signaling. While the role of dopamine in olfactory learning in Drosophila is well described, the function of serotonin and its downstream receptors on Drosophila olfactory learning remain largely unexplored. This study showed that the output of serotonergic neurons, possibly through points of synaptic contacts on the mushroom body (MB), is essential for training during olfactory associative learning in Drosophila larvae. Additionally, it was demonstrated that the regulation of olfactory associative learning by serotonin is mediated by its downstream receptor (d5-HT7) in a cAMP-dependent manner. d5-HT7 expression specifically in the MB, an anatomical structure essential for olfactory learning in Drosophila, is critical for olfactory associative learning. Importantly this work shows that spatio-temporal restriction of d5-HT7 expression to the MB is sufficient to rescue olfactory learning deficits in a d5-HT7 null larvae. In summary, these results establish a critical, and previously unknown, role of d5-HT7 in olfactory learning (Ganguly, 2020).

    Dopaminergic mechanism underlying reward-encoding of punishment omission during reversal learning in Drosophila

    Animals form and update learned associations between otherwise neutral sensory cues and aversive outcomes (i.e., punishment) to predict and avoid danger in changing environments. When a cue later occurs without punishment, this unexpected omission of aversive outcome is encoded as reward via activation of reward-encoding dopaminergic neurons. How such activation occurs remains unknown. Using real-time in vivo functional imaging, optogenetics, behavioral analysis and synaptic reconstruction from electron microscopy data, this study identified the neural circuit mechanism through which Drosophila reward-encoding dopaminergic neurons are activated when an olfactory cue is unexpectedly no longer paired with electric shock punishment. Reduced activation of punishment-encoding dopaminergic neurons relieves depression of olfactory synaptic inputs to cholinergic neurons. Synaptic excitation by these cholinergic neurons of reward-encoding dopaminergic neurons increases their odor response, thus decreasing aversiveness of the odor. These studies reveal how an excitatory cholinergic relay from punishment- to reward-encoding dopaminergic neurons encodes the absence of punishment as reward, revealing a general circuit motif for updating aversive memories that could be present in mammals (McCurdy, 2021).

    Associative learning in larval and adult Drosophila is impaired by the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine

    Across the animal kingdom, dopamine plays a crucial role in conferring reinforcement signals that teach animals about the causal structure of the world. In the fruit fly Drosophila melanogaster, dopaminergic reinforcement has largely been studied using genetics, whereas pharmacological approaches have received less attention. This study applied the dopamine-synthesis inhibitor 3-Iodo-L-tyrosine (3IY), which causes acute systemic inhibition of dopamine signaling, and investigated its effects on Pavlovian conditioning. 3IY feeding impairs sugar reward learning in larvae while leaving task-relevant behavioral faculties intact, and additional feeding of a precursor of dopamine (L-3,4-dihydroxyphenylalanine, L-DOPA), rescues this impairment. Concerning a different developmental stage and for the aversive valence domain, it was furthermore demonstrated that punishment learning by activating the dopaminergic neuron PPL1-γ1pedc in adult flies is also impaired by 3IY feeding and can likewise be rescued by L-DOPA. These findings exemplify the advantages of using a pharmacological approach in combination with the genetic techniques available in D. melanogaster to manipulate neuronal and behavioral function (Thoener, 2021).

    Dopamine-based mechanism for transient forgetting

    Active forgetting is an essential component of the memory management system of the brain. Forgetting can be permanent, in which prior memory is lost completely, or transient, in which memory exists in a temporary state of impaired retrieval. Temporary blocks on memory seem to be universal, and can disrupt an individual's plans, social interactions and ability to make rapid, flexible and appropriate choices. However, the neurobiological mechanisms that cause transient forgetting are unknown. This study identified a single dopamine neuron in Drosophila that mediates the memory suppression that results in transient forgetting. Artificially activating this neuron did not abolish the expression of long-term memory. Instead, it briefly suppressed memory retrieval, with the memory becoming accessible again over time. The dopamine neuron modulates memory retrieval by stimulating a unique dopamine receptor that is expressed in a restricted physical compartment of the axons of mushroom body neurons. This mechanism for transient forgetting is triggered by the presentation of interfering stimuli immediately before retrieval (Sadandal, 2021).

    Memory formation, consolidation and retrieval are well-known functions that support memory expression; however, the processes that limit these functions -- including forgetting -- are less understood. Forgetting has been characterized as either passive or active, and is crucial for memory removal, flexibility and updating. Memory may be removed completely, resulting in permanent forgetting; or temporarily irretrievable, resulting in transient forgetting (Sadandal, 2021).

    One form of active forgetting-known as intrinsic forgetting-involves one dopamine neuron (DAN) that innervates the γ2α'1 compartment of the axons of mushroom body neurons (MBNs) and the dendrites of the downstream, compartment-specific mushroom-body output neurons (MBONs). This DAN resides in a cluster of 12 DANs in each brain hemisphere that is known as the protocerebral posterior lateral 1 (PPL1) cluster. Current evidence indicates that the ongoing activity of these DANs after aversive olfactory conditioning slowly and chronically erodes labile and nonconsolidated behavioural memory, as well as a corresponding cellular memory trace that forms in the MBONs. This intrinsic forgetting mechanism is shaped by external sensory stimulation and sleep or rest, and is mediated by a signalling cascade in the MBNs that is initiated by the activation of the dopamine receptor DAMB, which leads to the downstream activation of the actin-binding protein Cofilin and the postulated reorganization of the synaptic cytoskeleton (Sadandal, 2021).

    By contrast, there is little understanding of the mechanisms that arbitrate transient forgetting. Neuropsychological studies of failures or delays in retrieval in humans have primarily focused on lexical access. Phonological blockers or interfering stimuli produce a tip-of-the-tongue state-the failure to recall the appropriate word or phrase. Tip-of-the-tongue states are resolved when the distracting signals dissipate. Several brain regions have been implicated in tip-of-the-tongue states from functional magnetic resonance imaging studies, but the neurobiological mechanisms that produce a temporary state of impaired retrieval are unknown. This study offers an entry point into this area of brain function (Sadandal, 2021).

    Memory retrieval has been proposed to consist of an interplay between internal or external cues and memory engrams, with cue-induced reactivation of engrams across multiple regions of the brain facilitating memory expression. But a central question about this process is how interfering stimuli temporarily block memory retrieval, resulting in transient forgetting. This study offers insights into one such mechanism. Behavioural and functional imaging data reveal that PPL1-α2α'2, working through the DAMB receptor expressed in the α2α'2 MBN axonal compartment, mediates the transient forgetting of PSD-LTM. This effect occurs without altering a cellular memory trace in the postsynaptic MBON-α2sc. This process can be triggered by distracting stimuli, illustrating a neural-genetic-environmental interplay that modifies memory expression (Sadandal, 2021).

    This study considered why the cellular memory trace remains unaffected by DAN stimulation despite the occurrence of behavioural forgetting. Because blocking synaptic output from MBON-α2sc reduces PSD-LTM expression, the simplest hypothesis posits that cellular memory traces form with conditioning in the MBON in addition to the cytoplasmic Ca2+-based memory trace that was detected in this study. This is expected: neurons undergo broad changes in physiology as they adopt new states, so it is plausible that such plastic mechanisms-especially ones that gate synaptic release-are inactivated by DAN activity while leaving the Ca2+-based memory trace intact (Sadandal, 2021).

    The discovery that loss of function of DAMB leads to enhanced PSD-LTM was surprising, because of a previous study reporting that this insult attenuates PSD-LTM. The experiments argue strongly that DAMB functions normally to suppress expression of PSD-LTM. However, this leads to the question of why a receptor involved in transient forgetting would lead to enhanced PSD-LTM when inactivated. Previous experiments have shown that PPL1-α2α'2-similar to PPL1-γ2α'1-exhibits ongoing activity, leading to a slow release of dopamine onto MBNs. This activity should slowly degrade or suppress existing memory so that when the receptor is inactivated memory expression is enhanced (Sadandal, 2021).

    PPL1-α2α'2 has no important role in the forgetting of labile nonconsolidated memory. Instead, previous studies have identified a different DAN (PPL1-γ2α'1) as having a role in this process and the apparent erasure of the downstream cellular memory trace-perhaps an indication of 'permanent forgetting'. This process is modulated by internal and external factors, and is mediated by key molecules expressed in the MBN that receive PPL1-γ2α'1 input. No robust decrement was found in expression of PSD-LTM after PPL1-γ2α'1 stimulation, which points to the existence of two separate dopamine-based circuits for permanent and transient forgetting. This functional separation may indicate a fundamental principle in the organization of circuits that mediate several forms of forgetting (Sadandal, 2021).

    However, the DAMB receptor is used for both permanent and transient forgetting. DAMB is widely expressed across the MBN axons but alters synaptic plasticity differently across MBN compartments. It is possible that DAMB signalling may be distinct for the two forms of forgetting. DAMB preferentially couples with Gq, the knockdown of which inhibits the potent erasure of memory, but its potential role in transient forgetting is unknown. The scaffolding protein Scribble orchestrates the activities of Rac, Pak and Cofilin, all of which are important for the permanent forgetting pathway. However, Scribble knockdown or inhibition of Rac1 does not enhance the PSD-LTM as is the case in DAMB-knockdown flies, which suggests that this scaffolding signalosome does not have a large role in transient forgetting. In summary, the two distinct forms of forgetting-transient and permanent-share a dopaminergic mechanism and a common dopamine receptor, but differ in upstream and downstream neural circuits and in downstream signalling pathways within MBNs (Sadandal, 2021)

    Transient Administration of Dopaminergic Precursor Causes Inheritable Overfeeding Behavior in Young Drosophila melanogaster Adults

    Imbalances in dopaminergic signaling during development have been indicated as part of the underlying neurobiology of several psychiatric illnesses. Yet, how transient manipulation of dopaminergic signaling influences long-lasting behavioral consequences, or if these modifications can induce inheritable traits, it is still not understood. This study used the Drosophila model to test if transient pharmacological activation of the dopaminergic system leads to modulations of feeding and locomotion in adult flies. Transient administration of a dopaminergic precursor, levodopa, at 6 h, 3 days or 5 days post-eclosion, induced overfeeding behavior, while no significant effects on locomotion were found. Moreover, this phenotype was inherited by the offspring of flies treated 6 h or 3 days post-eclosion, but not the offspring of those treated 5 days post-eclosion. These results indicate that transient alterations in dopaminergic signaling can produce behavioral alterations in adults, which can then be carried to descendants. These findings provide novel insights into the conditions in which environmental factors can produce transgenerational eating disorders (Moulin, 2020.

    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).

    Discrete serotonin systems mediate memory enhancement and escape latencies after unpredicted aversive experience in Drosophila place memory

    Feedback mechanisms in operant learning are critical for animals to increase reward or reduce punishment. However, not all conditions have a behavior that can readily resolve an event. Animals must then try out different behaviors to better their situation through outcome learning. Learned helplessness, as a type of outcome learning, manifests in part as increases in escape latency in the face of repeated unpredicted shocks. Little is known about the mechanisms of outcome learning. When fruit flies are exposed to unpredicted high temperatures in a place learning paradigm, flies both increase escape latencies and have a higher memory when given control of a place/temperature contingency. This study describes discrete serotonin neuronal circuits that mediate aversive reinforcement, escape latencies, and memory levels after place learning in the presence and absence of unexpected aversive events. The results show that two features of learned helplessness depend on the same modulatory system as aversive reinforcement. Moreover, changes in aversive reinforcement and escape latency depend on local neural circuit modulation, while memory enhancement requires larger modulation of multiple behavioral control circuits (Sitaraman, 2017).

    Dopamine receptor DAMB signals via Gq to mediate forgetting in Drosophila

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

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

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

    Cyclic AMP-dependent plasticity underlies rapid changes in odor coding associated with reward learning

    Learning and memory rely on dopamine and downstream cAMP-dependent plasticity across diverse organisms. Despite the central role of cAMP signaling, it is not known how cAMP-dependent plasticity drives coherent changes in neuronal physiology that encode the memory trace, or engram. In Drosophila, the mushroom body (MB) is critically involved in olfactory classical conditioning, and cAMP signaling molecules are necessary and sufficient for normal memory in intrinsic MB neurons. To evaluate the role of cAMP-dependent plasticity in learning, this study examined how cAMP manipulations and olfactory classical conditioning modulate olfactory responses in the MB with in vivo imaging. Elevating cAMP pharmacologically or optogenetically produced plasticity in MB neurons, altering their responses to odorants. Odor-evoked Ca(2+) responses showed net facilitation across anatomical regions. At the single-cell level, neurons exhibited heterogeneous responses to cAMP elevation, suggesting that cAMP drives plasticity to discrete subsets of MB neurons. Olfactory appetitive conditioning enhanced MB odor responses, mimicking the cAMP-dependent plasticity in directionality and magnitude. Elevating cAMP to equivalent levels as appetitive conditioning also produced plasticity, suggesting that the cAMP generated during conditioning affects odor-evoked responses in the MB. Finally, this plasticity was found to be dependent on the Rutabaga type I adenylyl cyclase, linking cAMP-dependent plasticity to behavioral modification. Overall, these data demonstrate that learning produces robust cAMP-dependent plasticity in intrinsic MB neurons, which is biased toward naturalistic reward learning. This suggests that cAMP signaling may serve to modulate intrinsic MB responses toward salient stimuli (Louis, 2018).

    Learning generates plasticity in neuronal responses to input stimuli, which is distributed across multiple cells and synapses in the brain. Molecularly, dopamine and downstream cAMP signaling are involved in multiple forms of memory, including olfactory learning. For instance, dopamine is required in the amygdala for olfactory classical conditioning in mammals. Similarly, dopamine and downstream cAMP signaling molecules play a central role in olfactory classical conditioning in Drosophila. This pathway is particularly critical in the mushroom body (MB), a brain region that receives olfactory information and is required for olfactory learning. Dopaminergic neurons are postulated to convey a reinforcement signal to the MB-stimulating certain subsets of MB-innervating dopaminergic neurons drives aversive or appetitive reinforcement in lieu of a physical reinforcer. The dopamine released from these neurons acts directly on intrinsic MB neurons, and possibly other neurons in the area as well. The D1-like receptor DopR, type I adenylyl cyclase Rutabaga (Rut), catalytic domain of protein kinase A, and Dunce phosphodiesterase (Dnc) are all required for olfactory classical conditioning. Importantly, rescuing the expression of DopR or Rut -- specifically in intrinsic MB neurons of otherwise mutant animals -- restores normal olfactory learning and memory. Further downstream, both Epac and PKA, as well as phosphorylation targets such as synapsin, have been shown to regulate learning and memory via effects in MB neurons. Thus, dopamine and cAMP are critical in intrinsic MB neurons for normal memory. Furthermore, broadly elevating cAMP generates plasticity in MB neurons, demonstrating that this pathway influences the responsivity of MB neurons. However, the role of this pathway in driving coherent patterns of plasticity that encode memory is unknown (Louis, 2018).

    Recent advances have opened up the possibility of understanding how olfactory memory is encoded in exquisite detail. Recent studies of memory encoding in the Drosophila MB have suggested that mushroom body neurons are highly plastic, exhibiting learning-related changes in odor responses. This is supported by observations of memory traces using in vivo Ca2+ imaging of neurons innervating the MB. However, the neuronal changes associated with cAMP-dependent, short-term memory are unclear. Conditioning generates plasticity in α'/β'-neurons within a few minutes of training, a time point at which the animals exhibit robust short-term memory. However, the Rut cyclase is not required in α'/β'-neurons for learning, leaving the functional role of cAMP-dependent plasticity in the MB unclear. MB γ-neurons exhibit depression in response to an aversive conditioned odor that is sensitive to manipulations of G αo-signaling, though it is not clear how this relates to dopaminergic modulation via G αs. Finally, blocking the synaptic output of MB neurons during conditioning does not impair aversive learning, suggesting that a significant proportion of the engram resides in the MB neurons and/or upstream connections (Louis, 2018).

    In contrast, other studies have described a major role for plasticity in downstream MB output neurons (MBONs), which may arise via pre- and/or postsynaptic plasticity. Robust, dopamine-dependent plasticity has been observed in MBONs, but not at the cellular level in MB neurons. This emphasizes the role of the MB in encoding sparse, relatively invariant olfactory representations. Learning-induced plasticity is then layered in at the MB-MBON synapses, possibly via synaptic depression. This leaves the requirement of cAMP signaling molecules in the MB, and the dispensability of MB output during memory acquisition, unresolved. Thus, there is a paradoxical dissociation of anatomical loci between where cAMP signaling is required and where robust, short-term, learning-induced plasticity has been reported. This study has examined the role of cAMP-dependent plasticity in the MB using in vivo imaging, combined with pharmacological and optogenetic manipulation of cAMP levels. Results suggest that cAMP-dependent plasticity localizes to intrinsic MB neurons and mirrors the plasticity induced during olfactory classical conditioning, with a bias toward appetitive conditioning (Louis, 2018).

    The present data support several major conclusions about the role of cAMP-dependent plasticity in the memory-encoding MB: (1) Intrinsic MB neurons exhibit robust cAMP-dependent plasticity; (2) cAMP-dependent plasticity is heterogeneous, both across and within anatomical classes of MB neurons; (3) the directionality and magnitude of plasticity parallel Rut-dependent associative changes in MB responsivity following appetitive classical conditioning; and (4) appetitive conditioning produces changes in cAMP of a magnitude that generates plasticity in odor-evoked responses. Thus, cAMP-dependent plasticity plays a major role in modulating intrinsic MB neurons, directly linking the physiology of MB neurons to the behavioral roles for cAMP signaling molecules in learning and memory. One caveat to the interpretation of imaging studies is that the preparations require tethering the animal under a microscope. Future developments enabling recording of brain activity in freely behaving animals will be necessary to test how responses in the MB neurons facilitate behavioral output in real time (Louis, 2018).

    In the context of olfactory learning, the MB encodes a sparse representation of olfactory space, which is computationally advantageous for learning and potentially modulated by learned valence. If neurons responded homogeneously to input stimuli, coincidence detection would result in uniform plasticity across the sparse set of neurons that encode the odor and receive a reinforcement signal. However, the heterogeneity observed in cAMP-dependent plasticity in this study suggests that olfactory memory traces may be driven to specific subsets of 'eligible' neurons in the MB. This could play an analogous role to memory allocation, which drives memory traces to subsets of eligible neurons in the mammalian amygdala during fear conditioning. Molecularly, heterogeneity may be driven by differential expression of genes that function downstream of cAMP/Epac/PKA to regulate neuronal excitability or presynaptic function. Such differences in expression could be set up via developmental or epigenetic mechanisms (Louis, 2018).

    It is proposed that the cAMP-dependent plasticity in the MB plays two roles during olfactory learning: filtering MB responses based on salience and encoding valence. A role for MB plasticity in salience filtering is suggested by the observation that appetitive conditioning produced enhancement of MB responses across spatial compartments. These compartments have been suggested to route olfactory signals to valence-encoding output neurons, driving learned approach or avoidance via heterosynaptic plasticity. Therefore, the broad pattern of plasticity observed in this study would affect multiple downstream output pathways of opposing valence, suggesting that it does not encode valence per se. Rather, it may function to heighten relative MB sensitivity to salient stimuli. Across multiple sensory systems, ascending information is filtered according to salience, typically enhancing responses to stimuli that are biologically important. Alternatively, appetitive conditioning may modulate MB neurons in a fundamentally different way from aversive conditioning. While these opposing forms of memory require many overlapping MB-associated neurons, there are some differences in the circuits recruited during these forms of learning, and plasticity across MB neurons may be one difference. Regardless of the interpretation, the data reveal cAMP-dependent plasticity at the cellular level in intrinsic MB neurons. This may be layered on top of synaptic plasticity at MB output synapses, which have been proposed to encode valence by altering how olfactory signals flow through the neuronal networks that mediate behavioral approach or avoidance (Louis, 2018).

    Several additional lines of evidence support the idea that cAMP-dependent plasticity serves as an overall gain control, regulating MB responses based on stimulus salience. First, the MB and MB-innervating dopaminergic neurons modulate salience-based decision making in a visual flight simulator paradigm. Second, dopaminergic neurons innervating the MB respond broadly to sensory stimuli that do not have an acquired valence, and exhibit activity that is correlated with locomotion. Activation of these neurons elevates cAMP in the downstream MB neurons in a compartmentalized manner, which in turn modulates their sensitivity and neurotransmission at the MB-MBON synapses. Thus, the MB neurons receive dynamically regulated dopaminergic inputs that alter the function of both the MB and downstream network components as a function of behavioral state. This may facilitate learning in situations in which the animal is likely to experience biologically important events (e.g., during foraging). Similar modulatory mechanisms modulate plasticity and memory in other animals as well. For instance, in honeybees, appetitive conditioning prolongs odor responses in MB neurons. Likewise, in the mammalian amygdala, coactivation of neuromodulatory and Hebbian plasticity is necessary for plasticity and memory (Louis, 2018).

    Aversive conditioning produced no significant plasticity in the current study, consistent with results from some optogenetic reinforcement substitution imaging experiments. However, since Rut is required in MB neurons for normal aversive memory, cAMP-dependent plasticity is likely present in some form. Indeed, a previous study detected plasticity in the γ-lobe following aversive conditioning, which could be tightly localized to specific output synapses or neuronal subsets. Pairing odor with stimulation of tyrosine hydroxylase Gal4-labeled dopaminergic neurons produces aversive memory and detectable plasticity in MB γ-neurons. In this study, robust plasticity differentially following appetitive conditioning. This may be due to a bias toward learning about stimuli that guide motivationally relevant behaviors, such as approaching food-associated odors. Consistent with such an interpretation, appetitive conditioning produces memory that is more stable over time than aversive memory. A single trial of appetitive conditioning leads to the formation of long-term memory, while aversive conditioning requires multiple-spaced trials. The cAMP-dependent plasticity during appetitive conditioning could trigger downstream molecular pathways necessary to engage long-term memory formation. This presumably interacts with Ca2+ levels in neurons to regulate short- and long-term memory. In honeybees, elevating intracellular Ca2+ during a single-trial conditioning, which normally only triggers short-term memory, can induce long-term memory, whereas decreasing intracellular Ca2+ during multiple-spaced training impaired long-term memory formation. In addition, appetitive memory retrieval is motivationally gated by hunger state, suggesting a tie-in with motivational state. Integrating these observations, this suggests that motivationally relevant stimuli may enhance the sensitivity of MB neurons via cAMP-dependent plasticity, modulating the overall gain of the system in a salience-dependent manner (Louis, 2018).

    The MB is involved in multiple distinct yet potentially interrelated behaviors, including several forms of learning and memory, regulating sleep and activity, context generalization, habituation, temperature preference, context dependence of olfactory behaviors, and salience-based decision making. The common thread among these behaviors is that they revolve around selection of an appropriate action based on context. Thus, a primary function of the MB and its modulatory input may to be alter the probability of action based on integrating environmental cues and internal state. In such a scenario, modulating the overall gain of the circuit could function in concert with fine-scale synapse-specific plasticity to alter the flow of information to downstream motor areas. Thus, these data support a model in which dopaminergic neurons and downstream cAMP-dependent plasticity modulate MB responses to stimuli based on their salience, priming the animal to engage in appropriate goal-oriented behaviors (Louis, 2018).

    The neural circuit linking mushroom body parallel circuits induces memory consolidation in Drosophila

    Memory consolidation is augmented by repeated learning following rest intervals, which is known as the spacing effect. Although the spacing effect has been associated with cumulative cellular responses in the neurons engaged in memory, this study reports the neural circuit-based mechanism for generating the spacing effect in the memory-related mushroom body (MB) parallel circuits in Drosophila. To investigate the neurons activated during the training, expression was monitored of phosphorylation of mitogen-activated protein kinase (MAPK), ERK [phosphorylation of extracellular signal-related kinase (pERK)]. In an olfactory spaced training paradigm, pERK expression in one of the parallel circuits, consisting of gammam neurons, was progressively inhibited via dopamine. This inhibition resulted in reduced pERK expression in a postsynaptic GABAergic neuron that, in turn, led to an increase in pERK expression in a dopaminergic neuron specifically in the later session during spaced training, suggesting that disinhibition of the dopaminergic neuron occurs during spaced training. The dopaminergic neuron was significant for gene expression in the different MB parallel circuits consisting of alpha/betas neurons for memory consolidation. These results suggest that the spacing effect-generating neurons and the neurons engaged in memory reside in the distinct MB parallel circuits and that the spacing effect can be a consequence of evolved neural circuit architecture (Awata, 2019).

    A connectome of a learning and memory center in the adult Drosophila brain

    Understanding memory formation, storage and retrieval requires knowledge of the underlying neuronal circuits. In Drosophila, the mushroom body (MB) is the major site of associative learning. This study reconstructed the morphologies and synaptic connections of all 983 neurons within the three functional units, or compartments, that compose the adult MB's alpha lobe, using a dataset of isotropic 8 nm voxels collected by focused ion-beam milling scanning electron microscopy. It was found that Kenyon cells (KCs), whose sparse activity encodes sensory information, each make multiple en passant synapses to MB output neurons (MBONs) in each compartment. Some MBONs have inputs from all KCs, while others differentially sample sensory modalities. Only 6% of KC>MBON synapses receive a direct synapse from a dopaminergic neuron (DAN). Two unanticipated classes of synapses, KC>DAN and DAN>MBON, were identified. DAN activation produces a slow depolarization of the MBON in these DAN>MBON synapses and can weaken memory recall (Takemura, 2017).

    Associative memory helps animals adapt their behaviors to a dynamically changing world. The molecular mechanisms of memory formation are thought to involve persistent changes in the efficiency of synaptic transmission between neurons. In associative learning, persistent changes in synaptic efficacy correlated with memory formation have been found at points of convergence between two neuronal representations: one providing information from sensory inputs about the outside world and a second indicating whether the current environment is punitive or rewarding. Such sites of convergence have been identified for multiple forms of associative learning. However, a comprehensive synaptic level description of connectivity at such a site of convergence is not available for an animal as complex as the fruit fly, Drosophila (Takemura, 2017).

    The mushroom body (MB) is the center of associative learning in insects. Sensory information enters the MB via the calyx, where the dendritic claws of Kenyon cells (KCs) receive synaptic inputs from projection neurons of olfactory and other modalities including visual, gustatory and thermal. The parallel axonal fibers of the KCs form the MB-lobes, the output region of the MB. A pattern of sparse activity in the KC population represents the identity of the stimulus. This sparseness is maintained through two mechanisms. First, individual KCs generally only spike when they receive simultaneous inputs from multiple projection neurons. Second, overall KC excitability is regulated by feedback inhibition from a GABAergic neuron, MB-APL, that arborizes throughout the MB. Thus, only a small subset of KCs respond to a given sensory stimulus. Upon this representation of the sensory world, dopaminergic or octopaminergic neurons convey information of punishment or reward and induce memories that associate the sensory stimulus with its valence (Takemura, 2017).

    The functional architecture of the MB circuit is best understood in adult Drosophila (see Diagram of the α lobe of the mushroom body). In each MB, the parallel axonal fibers of ~2000 KCs can be divided into 16 compartmental units by the dendrites of 21 types of MB output neurons (MBONs) and the axon terminals of 20 types of dopaminergic neurons (DANs). A large body of behavioral and physiological studies suggests that these anatomical compartments are also parallel units of associative learning (see e.g., Hige, 2015; Lin, 2014). In each compartment, the dendrites of a few MBONs overlap with axon bundles of hundreds of KCs. Punishment and reward activate distinct sets of DANs. DAN input to a compartment has been shown to induce enduring changes in efficacy of KC>MBONs synapses in those specific KCs that were active in that compartment at the time of dopamine release. The valence of the memory appears to be determined by which compartment receives dopamine during training, while the sensory specificity of the memory is determined by which KCs were active during training (Takemura, 2017).

    Compartments can have distinct rates of memory acquisition and decay, and the 16 compartments together appear to form a set of parallel memory units whose activities are coordinated through both direct and indirect inter-compartmental connections. The DANs which project to the α1 compartment, the ventral-most compartment of the vertical lobe, play a key role in the formation of appetitive long-term memory of nutritional foods. DANs that project to the other α lobe compartments, α2 and α3, play roles in aversive long-term memory. All three of these compartments receive feedforward inputs from GABAergic and glutamatergic MBONs whose dendrites lie in other MB compartments known to be involved in aversive or appetitive memory. In addition, two types of MB-intrinsic neurons send arbors throughout the MB-lobes: a large GABAergic neuron, MB-APL, which provides negative feedback important for sparse coding, and the MB-DPM neuron, which is involved in memory consolidation and sleep regulation (Takemura, 2017).

    Previous EM studies in the MB lobes of cockroaches, locusts, crickets, ants, honey bees and Drosophila identified KCs by their abundance, fasciculating axons and small size. Additionally, large GABA immunoreactive neurons that contact KC axons were identified in the locust pedunculus. While these data provided early insights to guide modeling of the MB circuit, the volumes analyzed were limited and most neuronal processes could not be definitively assigned to specific cell types. This paper reports a dense reconstruction of the three compartments that make up the α lobe of an adult Drosophila male. Because a dense reconstruction was performed, with the goal of determining the morphology and connectivity of all cells in the volume, it can be confidently stated that all cell types with processes in the α lobe have been identified (Takemura, 2017).

    Comprehensive knowledge of the connectivity in the α lobe has allowed addressing of several outstanding issues. The first concerns the nature of KC>MBON connectivity. Although each KC passes through all three compartments, it is not known if individual KCs have en passant synapses in each compartment. Thus, it remains an open question whether the sensory representation provided to each compartment and each MBON within a compartment is the same or whether different MBONs within a compartment might sample from non-overlapping sets of KCs, and thus use independent sensory representations for learning. It was also not known which, if any, other cell types are direct postsynaptic targets of KCs (Takemura, 2017).

    The second concerns dopamine modulation. What are the locations of dopaminergic synapses and what does this distribution imply about the targets of dopaminergic modulation as well as volume versus local transmission? Cell-type-specific rescue of dopamine receptor mutants suggests that dopamine acts presynaptically in the KCs of KC>MBON synapses. However, postsynaptic mechanisms have also been proposed and a recent study detected expression of dopamine receptors in MBONs, raising the possibility that MBONs might also be direct targets of DAN modulation. Behavioral, imaging and electrophysiological data indicate that dopamine modulation respects the borders between compartments, but it is not known whether these borders have a distinct structure, such as a glial sheet (Takemura, 2017).

    The third concerns the two MBON types that send feedforward projections into the α lobe. These MBONs have important roles in associative learning as revealed by behavioral assays and have been postulated to integrate memories of opposing valence and different time scales. However, it is not known which cell types these feedforward MBON projections targets within the MB (Takemura, 2017).

    The fourth concerns the two neurons, MB-APL and MB-DPM, which arborize throughout the MB and are thought to regulate MB function globally. What is their local synaptic connectivity within the α lobe and what can this inform about how they perform their roles (Takemura, 2017)?

    Finally, the three compartments of the α lobe differ in important aspects, including valence of the memory formed, the time course of memory formation and retrieval, and the numerical complexity of their DAN inputs and MBON outputs. Are there obvious differences in the microcircuits of different compartments (Takemura, 2017)?

    In this paper reports the answers to these questions. In addition, the utility of detailed anatomy at the electron microscopic level to provide novel insights is demonstrated: It is shown that nearly all cell types in the α lobe contain more than one morphological class of synaptic vesicle, raising the possibility that these cells utilize multiple neurotransmitters. In addition, two prevalent sets of synaptic motifs - from DANs to MBONs and from KCs to DAN s- are described that were unanticipated despite the extensive anatomical, physiological, behavioral and theoretical studies that have been performed on the insect MB. These novel DAN to MBON connections are characterized using behavioral and physiological assays and find that DAN activation produces a slow depolarization of postsynaptic MBONs and can weaken memory recall (Takemura, 2017).

    The connections between the neurons observed in this study are are summarized in a Summary diagram of the connectome reconstruction of the α lobe. In each of the lobe's three compartments, parallel axonal fibers of ~1000 KCs project through the dendrites of a few MBONs and the terminal arbors of a few DANs. The results provide support for several aspects of the generally accepted model for MB circuit function. First, it was found that each KC forms en passant synapses with multiple MBONs down the length of its axon, making it possible for parallel processing across the different compartments of the MB lobes. Secondly, with the assumption that released dopamine diffuses locally, KC>MBON synapses would receive dopaminergic input close to the sites of vesicle release, consistent with the prevailing hypothesis that plasticity occurs at the presynaptic terminals of KCs. However, several circuit motifs were found that were not anticipated by previous work. For example, synaptic connections were found from KCs to DANs, indicating that DANs get axo-axonal inputs within the MB lobes themselves. A recent report provides evidence that these KC>DAN synapses are functional (Cervantes-Sandoval, 2017). An even more unexpected motif was the direct synaptic contacts from DAN to MBON found in every compartment. Functional connectivity experiments confirmed that these connections are monosynaptic, and showed that they give rise to a slow depolarization in the MBON. Moreover, stimulating DANs in freely behaving flies yields effects consistent with a net excitatory DAN>MBON connection. Finally, the synaptic connections are described of two feedforward MBONs, which have been proposed to mediate the interaction of the various parallel memories within the MB lobes, as well as two intrinsic MB neurons, APL and DPM (Takemura, 2017).

    This work not only provides definitive evidence for, and quantitative detail about, many previously observed circuit motifs, but also reveals several motifs not anticipated by prior anatomical, behavioral or theoretical studies. These additional circuit motifs provide new insights and raise new questions about the computations carried out by the MB. It is noted that these same novel connections were also found in a parallel study of the larval MB (Eichler, 2017). Not only were the same circuit motifs found in the larval MB and adult α lobe, but also the relative prevalence of these connections was strikingly similar: DAN>MBON synapses were 4.5% the number of KC>MBON synapses in the adult α lobe and 3.4% in the larval MB. KC>DAN synapses were 1.5 times as prevalent as DAN>KC synapses in the adult α lobe, as compared with 1.1 in the larval MB. KCs make 48% of their synapses onto other KCs in the adult α lobe and 45% in the larval upper vertical lobe compartments. It is tempting to speculate that the conservation of the relative abundances of these connections across developmental stages reflects important functional constraints on the circuit (Takemura, 2017).

    A large body of work supports the idea that individual KC>MBON synapses are the elemental substrates of associative memory storage in the MB. The dominant hypothesis in the field is that coincidence detection occurs within the presynaptic terminals of the KCs. The Conditioned Stimulus (CS, for example an odor) evokes a spiking response in a sparse subset of KCs, which in turn leads to Ca2+ influx. The Unconditioned Stimulus (US, for example electric shock) activates dopaminergic inputs to the MB lobes, where they likely activate G-protein-coupled dopamine receptors on the KC cell membrane. The coincidence of these two events is thought to be detected by the Ca2+ sensitive, calmodulin-dependent adenylate cyclase rutabaga, which initiates a cAMP signaling cascade that leads to the biochemical changes underlying synaptic plasticity (Takemura, 2017).

    The tiling of MBON and DAN projections down the length of the KC axons suggests that each of these compartments serves as an independent module, with the association of reinforcement with sensory input taking place in parallel across several different modules. One important assumption in this model is that each KC sends parallel input to each compartment by making synapses all the way down the length of its axon. Light microscopic imaging established that the axons of individual α/β KCs do indeed run through all three compartments of the α lobe. However, they also revealed that the axonal branching patterns differ between KC classes. For example, the axons of α/βp KCs branch in α2, whereas those of α/βc and α/βs KCs do not, raising the question of how extensive KC outputs are across the different compartments. The dense EM reconstruction established that in fact all α/β KCs form en passant synapses on MBONs in each of the three α lobe compartments (Takemura, 2017).

    In many cases, these synapses were found at enlarged boutons that contained the presynaptic machinery. However, output sites were also found on the smooth axons of the α/βc KCs, which lack obvious bouton-like swellings. Only occasional, short (generally <5 μm) segments of KC axons where the axon became thinner than 300 nm in diameter lacked presynaptic sites. Of course, it is not known whether all these synapses are functional. EM analysis showed that within each compartment, every KC passing through a layer of the compartment that was extensively innervated by an MBON made at least one synapse with that MBON. Previous electrophysiological measurements of connectivity in the α2 compartment indicated that only about 30% of KCs connect to MBON-α2sc, suggesting the possibility that the majority of KC>MBON synapses are functionally silent, as they are in cerebellar cortex, where 98% of the parallel fiber-to-Purkinje cell synapses are believed to be silent. However, a more trivial explanation cannot be ruled out: These measurements were made in the presence of cholinergic antagonists that could have partially blocked synaptic events and lead to an underestimate of total connectivity levels (Takemura, 2017).

    The EM data revealed that the the number of synapses made by individual KCs was well-described by a Poisson distribution, where each synapse connects with a uniform, independent, and random probability to one of the KCs. Although the predicted distributions strongly depend on the number of connections between two cell types, almost all KC connections to other cells obeyed Poisson statistics. This was true of every KC in the α1 and α3 compartments, where each MBON has compartment-filling dendrites. The α2 compartment is somewhat unusual in that its MBONs innervate only subzones of the compartment. While light microscopy showed that MBON-α2sc primarily innervates the surface and core of the compartment, MBON-α2sp was found to project more to the surface and posterior. The connectome results bore out these observations from the light and electron microscopy, although EM reconstructions also showed that these borders were not sharp, and these MBONs receive less extensive and weaker connections outside these subzones. Nevertheless, within the primary area of innervation, it was again the case that every KC made synapses with all MBONs along its axon. Thus each of the 949 α/β KCs can deliver information to the MBONs in each of the three α lobe compartments (Takemura, 2017).

    A strictly feed-forward view of the circuit may miss important processing, however, as earlier studies suggested, and the current results re-emphasize. Firstly, gap junctions between KCs have been reported. This opens up the possibility for lateral propagation of signals across KCs, either biochemical or electrical. For example, in mammalian systems, axo-axonal gap junction coupling can synchronize firing between neurons. Secondly, chemical synapses between KCs have been reported in the MB pedunculus in the locust. The reconstructions show that such KC>KC connections are also present in the lobes, where they are surprisingly prevalent. In fact, the most frequent outputs of the α/βs KCs are other α/βs KCs, assuming the morphologically defined KC>KC connections are functional synapses (Takemura, 2017).

    A high percentage (55%) of these putative KC>KC synapses occur in rosette-like structures where multiple KCs also converge on a single dendritic process of an MBON . These are relatively unusual structures, not observed in EM reconstructions of the Drosophila visual system and, indeed, there is no direct evidence that they are functional synapses. At present it is only possible to speculate on their role. As points of heavy convergence, they might allow the effects of synapses from different KCs onto the same dendrite to act synergistically. Activity of a single KC may spread to its neighbors within the rosette, potentially generating a large compound synaptic release event onto the MBON in the middle. Such a signal amplification mechanism may be important to ensure that individual KCs can have a significant impact on MBON membrane potential by recruiting their rosette partners. How the specificity of learning could be maintained in this scenario is, however, unclear. Several basic questions will need to be answered before it is possible to begin to understand the functional significance of these rosettes. For example, can a single KC in the rosette indeed activate its neighbors? And how similar are the response properties of the different KCs that contribute to one rosette (Takemura, 2017)?

    In conclusion, the connectivity of the KCs that carry olfactory and other sensory representations supports a model where parallel distributed memory processing occurs in each compartment. However, several circuit motifs that seem designed to spread and possibly amplify signals at the sites of KC output indicate that this circuit is likely more complicated than a simple feed-forward view of the system suggests (Takemura, 2017).

    Dopamine-induced plasticity of the KC>MBON synapse is thought to be central to associative learning in this system. The reconstructions showed that dopaminergic neurons make well-defined synaptic contacts within the α lobe, with closely apposed post-synaptic membranes. This contrasts somewhat with dopaminergic innervation in the mammalian system, where there is typically not such close contact with a single clear post-synaptic partner, and volume transmission is the predominant model for dopamine release. It is not known whether the direct and indirect dopaminergic release sites have different functional consequences. Nevertheless, it seems likely that some type of volume transmission happens in the mushroom body. First, ~10 times more KC>MBON synapses than presynaptic sites of dopamine release were found in the α lobe, but previous work showed that learning-induced plasticity depresses MBON responses so strongly that most inputs are likely affected. Second, dopamine would need to diffuse only ~2 μm to reach every KC>MBON synapse within a compartment, but would also be sufficiently short range to prevent significant spill-over of dopamine to neighboring compartments, ensuring that the modularity of plasticity is maintained (Takemura, 2017).

    Functional connectivity measurements showed that stimulating the DANs elicits large amplitude calcium signals from MBONs, similar to previous results. Intracellular recordings revealed that this was a surprisingly strong connection, sufficient to elicit spikes in the MBON. The response persisted when both spiking and nicotinic transmission was blocked, to limit the possibility that the DANs act through the KCs, which are cholinergic. Conversely, the response was strongly reduced by adding a dopamine receptor antagonist. Taken together, these results indicate that the response is likely a direct action of dopamine released by the DANs on the MBON, although a more complex mechanism or a role for the transmitter contained in the dense core vesicles observed in the DANs cannot be formally rule out. The depolarization exhibited markedly slow dynamics, peaking >2 s after stimulation offset, and then decaying over tens of seconds. Dopaminergic responses of similar amplitude and time course have been reported in both mammalian systems and in Aplysia, where it is mediated by cAMP-driven changes in a non-selective cation conductance (Takemura, 2017).

    It is possible to induce memory formation in this circuit by pairing odor delivery with artificial activation of DANs. Targeting this optogenetic training procedure to DANs that innervate different compartments within the α lobe gives rise to memories with different valence, induction threshold and persistence. In the α1 compartment, a single pairing for 1 min induces an appetitive memory that lasts for 1 day. In contrast, optogenetic training focused on the α3 compartment requires multiple 1 min pairings, repeated at spaced intervals, and induces an aversive memory that lasts for 4 days. Although it seems likely that the different valences reflect the different projection sites of the MBONs for each of these compartments, where the differences in induction threshold and memory persistence might arise is less clear. There is no simple explanation for these differences from the EM-level circuit structure, as the basic wiring motifs were very similar in each compartment. Moreover, any explanation that invokes biochemical differences in KC>MBON synapses would require crisp spatial localization of the signaling pathway machinery that triggers plasticity, as exactly the same KCs participate in memory formation in different compartments. However, the observation that there are DAN>MBON synapses raises the possibility that biochemical differences in the MBONs might contribute to these differences in plasticity induction and maintenance. Indeed, RNAseq data from a set of four different MBONs showed expression of dopamine receptors. An alternative possibility, suggested by the findings in this study, is that the cotransmitter found in the dense core vesicles in the DANs is responsible for these differences. The size of these vesicles differs between DANs innervating the different compartments. Thus, these cells might release distinct co-transmitters, as has been observed in mammalian brain, which could trigger different signaling cascades in either the KCs or the MBONs to differentially modulate the induction and expression of plasticity across compartments (Takemura, 2017).

    Models of MB function have generally considered the role for DANs to be confined to relaying signals about punishment or reward to the MB. However, in the mammalian brain, DANs can dynamically change their responses to both US and CS. In this study, it was found that the axonal terminals of the DANs receive many inputs from KCs within the lobes. In other words, both MBONs, DANs and even KCs receive extensive synaptic input from KCs in each compartment. If the current model that plasticity is pre-synaptic proves to be correct, this suggests that the responses of the DANs themselves would be subject to plasticity. If the synaptic depression observed at KC>MBON synapses also acts at KC>DAN connections, odor-evoked DAN responses would be diminished as a result of learning. This would serve as a negative feedback loop, reducing the strength of plasticity on successive training cycles with the same odor. Indeed, a gradually plateauing of the learning curve is a common feature of memory formation in different systems, including olfactory conditioning in Drosophila (Takemura, 2017).

    One of the more surprising findings of this study was the observation that there are many direct DAN>MBON synaptic connections. Moreover, the functional connectivity measures indicate that these were relatively strong excitatory inputs. The excitatory sign of the DAN>MBON connection is also consistent with the behavioral effects of DAN activation that was observed. What role these DAN>MBON connections play in overall circuit function is an important question for future work. There are two general possibilities that are felt to be interesting to consider. Dopaminergic modulation has been proposed to play a general role in routing of information through the MB to different downstream neurons. Although changes in KC>MBON strength contribute to this process, the current results suggest that such state changes could also potentially be conveyed to the MBONs directly from the DANs. State-dependent changes in DAN activity have indeed been observed with calcium imaging. The slow synaptic dynamics observed in the DAN>MBON connection in MBON-α1 suggest the possibility that small changes in DAN firing might be capable of producing sustained changes in MBON membrane potential reflecting the current internal state of the animal (Takemura, 2017).

    A second possibility, suggested from the framework of reinforcement learning established in vertebrates, is related to motivation and the comparison of expected versus actual reward. In Drosophila, prior work on odor-sugar conditioning in larvae provided evidence that flies form a comparison between the current state of reward and the reward expected from the conditioned cue. This work showed that animals behaviorally express memories only when the expected reward intensity is higher than the currently available reward. This is similar to the results presented in this study; just as the presence of reward diminished memory expression in the larvae, stimulating the DANs suppressed performance of animals trained by the optogenetic conditioning. The need to compare current and expected reward could potentially explain why there is an opponent relationship between the depression of KC>MBON synapses that drives associative learning and the excitatory effects of the DAN>MBON connection. If depression dominates, the association drives behavior, but this can be overridden by sufficient levels of DAN activity. In this respect, it is noteworthy that DANs appear to be able to act directly on the MBON, without participation of the KCs. Overall, this comparison could ensure that learned behavior is motivated not strictly by the expectation of reward, but rather the expected increase in reward, assessed at the moment of testing (Takemura, 2017).

    The organization of the MB into a set of compartments arranged in series along the KC axons is well suited for simultaneously storing multiple independent memories of a given sensory stimulus. However, there must be some means by which these modules interact with one another to ensure coordinated, coherent expression of memory. Feedforward connections that link different compartments, first discovered by light microscopic anatomy, have recently been shown to be important for mediating such interactions. In particular, MBON-γ1pedc>α/β is an inhibitory neuron that connects aversive and appetitive learning compartments; it ensures that the circuit can readily toggle between different behavioral outputs (Takemura, 2017).

    The EM reconstructions included both MBON-γ1pedc>α/β and MBON-β1>α, two feedforward neurons which project from their respective compartments to widely innervate other parts of the MB. Memories stored in the α lobe compartments are long-term and relatively inflexible, whereas the short-term memories formed in β1 and γ1pedc are readily updated by recent experiences. The feedforward connections are thought to enable the short-term memories in β1 and γ1pedc to temporarily mask expression of the stable memories stored in the α lobe. Indeed training an animal with either a multi-component aversive/appetitive food stimulus, or by simultaneous optogenetic activation of a composite set of DANs covering both appetitive and aversive compartments results in a compound memory that is initially aversive and later transitions to appetitive. The connectome results show that the primary synaptic targets of these feedforward neurons are the MBONs in the downstream compartment. By contrast, relatively few connections onto KCs were observed. Overall, this suggests that the feedforward connections can strongly influence the output from a compartment, but likely have little impact on the sensory information delivered to each compartment from the KCs. This is consistent with observations that MBON-γ1pedc>α/β strongly modulates activity of glutamatergic neurons at the tip of the horizontal lobe, but not their dendritic responses. Targeting these feedforward connections to the MBON may ensure that conflicting memories can form simultaneously in response to a complex sensory input, but with the behavioral manifestation of those memories capable of undergoing a crisp switch (Takemura, 2017).

    This study has provided synapse level anatomical information on neuronal circuits involved in learning and memory in Drosophila. The comprehensive nature of this dataset should enable modeling studies not previously possible and suggests many experiments to explore the physiological and behavioral significance of the circuit motifs that were observed. That many of these motifs were not anticipated by over 30 years of extensive anatomical, experimental and theoretical studies on the role of the insect MB argues strongly for the value of electron microscopic connectomic studies (Takemura, 2017).

    A dense (complete) reconstruction of neurons and synapses is resource intensive, so it is reasonable to ask if tracing a subset of cells or synapses could have yielded similar results with less effort. This is hard to answer in general, since there are many sparse tracing strategies, and each can be pursued to differing degrees of completeness. It is likely that most sparse tracing strategies would have discovered the new pathways reported in this, as the connections are numerous and connect well known cell types. Conversely, the conclusions that all cell types in this circuit had been identified would have been more difficult to make with confidence and a rare cell type, such as the SIFamide neuron, might have been missed. Perhaps, most importantly, statistical arguments, particularly those that require an accurate assessment of which cells are not connected, such as the absence of network structures such as rings or chains, would have been hard to make from sparse tracing. More generally, the model independent nature of dense tracing helps to discover any 'unknown unknowns', provides the strongest constraints on how neural circuits are constructed, and allows retrospective analysis of network properties not targeted during reconstruction (Takemura, 2017).

    Developmental analysis of the dopamine-containing neurons of the Drosophila brain

    The Drosophila dopaminergic (DAergic) system consists of a relatively small number of neurons clustered throughout the brain and ventral nerve cord. Previous work shows that clusters of DA neurons innervate different brain compartments, which in part accounts for functional diversity of the DA system. This study analyzed the association between DA neuron clusters and specific brain lineages, developmental and structural units of the Drosophila brain that provide a framework of connections that can be followed throughout development. The hatching larval brain contains six groups of primary DA neurons (born in the embryo), which are assigned to six distinct lineages. All larval DA clusters persist into the adult brain. Some clusters increase in cell number during late larval stages, whereas others do not become DA positive until early pupa. Ablating neuroblasts with hydroxyurea (HU) prior to onset of larval proliferation (generates secondary neurons) confirms that these added DA clusters are primary neurons born in the embryo, rather than secondary neurons. A single cluster that becomes DA positive in the late pupa, PAM1/lineage DALcm1/2, forms part of a secondary lineage that can be ablated by larval HU application. By supplying lineage information for each DA cluster, this analysis promotes further developmental and functional analyses of this important system of neurons (Hartenstein, 2017).

    DA neurons of the early and late larval brain were labeled by anti-TH and TH-Gal4 driving UAS-mCD8::GFP. Both markers, used in the same brain specimen, labeled identical clusters of neurons, confirming previous studies thet concluded that the expression of TH and DA 'probably label the same neurons.' Neurons labeled by anti-DA or anti-TH/TH-Gal4-positive neurons can be expected to contain DA, even though that in itself is admittedly no proof that they are dopaminergic, in the sense that they utilize dopamine as a transmitter. That being said, this study will in the following refer to these neurons as 'DA neurons', following the convention established in the recent literature. The pattern was compared of DA clusters in the early larva (L1) and late larva (L3). The DA population comprises two dorsomedial clusters (DM1/2) and two lateral clusters (DL1/2). DM1 and DL2 are split into two smaller units each (DM1a/b and DL2a/b, respectively) (see Lineage identity of larval DA neurons). Since most larval DA neurons appear as differentiated cells already in L1, they constitute embryonically born primary neurons, a supposition confirmed by the analysis of Blanco (2011) who could assign the seven clusters to seven separate neuroblast clones induced in the embryo. One cluster, DM2, is not yet represented at L1; cells of this cluster gradually express TH-Gal4 during the L3 stage. In mid-L3 larvae, DM2 is typically represented by a single TH-Gal4-positive neuron; by late L3 the number has increased to about 6. It is concluded that, even though all DA neurons are born in the embryo, they initiated TH expression at different stages in the larva. As shown below, several groups of primary neurons remain TH-Gal4-negative throughout the larva and initiate expression during metamorphosis (Hartenstein, 2017).

    To determine the specific lineage identity of the DA clusters, the trajectory of their primary axon tracts was mapped relative to the secondary tracts in the late larval brain, for which a complete lineage atlas has been established. The underlying assumption is that the axon tracts of secondary lineages fasciculate with, or are at least are very close to, the axon tracts formed by primary neurons. Such a spatial relationship has been shown for many lineages for which both primary and secondary components were labeled together. To visualize the secondary axon tracts (SATs), brains were labeled with anti-Neurotactin (Nrt). It is apparent that most of the GFP-positive axons of DA neurons fasciculate with specific SATs, which was then taken as the criterion to assign a given DA neuron to the corresponding lineage (Hartenstein, 2017). The first objective of this paper was to assign the brain DA neurons to neural stem cell lineages and follow them from their time of appearance to the adult stage. At least six different lineages contain primary neurons that express TH during the larval stage. Primary neurons of four additional lineages initiate TH expression past the onset of metamorphosis. The second goal was to establish whether primary DA neurons visible in the larva are maintained in the adult, or are replaced by secondary neurons. The former is the case, as shown by the continued presence of labeled (antibody or TH-Gal4> UAS-mCD8::GFP) clusters of neurons at corresponding positions at closely spaced pupal stages, and the fact that application of HU during larval stages does not eliminate DA neurons in the adult. Only one group of DA neurons, PAM1, consists of secondary neurons of lineage DALcm1/2, born in the larva, and differentiating during late pupal stages. Interestingly, a recent study (Rohwedder, 2015) in which a different antibody directed against TH was used, discovered four additional primary neurons (pPAM) that express DA, and that, based on their branching pattern (around the medial lobe of the mushroom body) belong to the lineage DALcm1/2 (Hartenstein, 2017).

    Projections of the different groups/lineages of DA neurons can be assigned to different compartments of the brain. Notably, PPL1 (CP2/3 lineage) innervates the lobes of the mushroom body and the adjacent IPa compartment in the anterior protocerebrum; PPM3 (CM4 lineage) branches widely in the different compartments of the central complex and adjacent LAL, and PPL2 (BLVa1/2) projects to the posterior SLP. Within a given group, DA neurons differ further in regard to their exact branching pattern. PPL1 neurons fall into five different types innervating different domains within the vertical lobe. PPM3 neurons project to different compartments of the central complex; for example, one subset innervates the upper layers of the fan shaped body (FB) and the ellipsoid body (EB); another one is restricted to the lower FB and noduli (NO). These findings suggest that primary neurons of the CP2/3 lineage have a unique identity, which coincides with numerous previous studies of primary neurons belonging to other lineages (Hartenstein, 2017).

    At present, not much information is available about the primary lineages that include the clusters of DA neurons of the larval brain. Primary neurons are produced in the embryo by the proliferatory activity of approximately 100 pairs of neuroblasts. These cells undergo 5-8 rounds of division, producing lineages of 10-16 cells each. Approximately 30% of primary neurons undergo programmed cell death at the embryo-larva transition, reducing the average size per larval primary lineage to about 10. Still, this number exceeds the number of DA neurons in any of the clusters in the larval brain, implying that not all cells of a given lineage express the DA phenotype. Future studies, employing lineage-specific markers, are required to establish these additional details about the DA neuronal genealogy (Hartenstein, 2017).

    Location and projection pattern of DA neurons have been mapped for numerous insect species, among them locusts and bees. In all species, DA neurons are distributed in discrete clusters, scattered over much of the brain surface, suggesting that as in Drosophila, these neurons form part of multiple lineages. In some cases, it is evident that lineages producing DA neurons are homologous. For example, the C1/C2 clusters of DA neurons in the bee brain (Schäfer and Rehder, 1989) quite clearly correspond in cell body location and projection (to the lobes of the mushroom body) to PAM1/DALcm1/2. Likewise, C3 of the bee may correspond to a combination of PPL1/CP and PPM3/CM4. C3 emits two separate fiber bundles, one of which projects and ramifies towards the central complex; this could represent the PPM3/CM4 group in fly which projects via the MEF fascicle to the central complex. The second contingent of bee C3 DA neurons projects towards the lobes of the mushroom body in the anterior neuropil; this group may correspond to PPL1/CP in Drosophila. Aside from the above mentioned similarities, one very obvious difference exists in the antennal lobe with regards to in the distribution of DA neurons and their terminal arbors. In Drosophila, only two neurons which start expressing TH during metamorphosis are associated with this compartment, whereas in other insects (like bee), the antennal lobe receives dense projections from DA neurons located adjacent to it. It will be an interesting and rewarding task to establish, lineage by lineage, the similarities and differences in the anatomy of the DA system (as well as other groups of neurons sharing similar neurotransmitter expression patterns) among different insect taxa, and to correlate these patterns with physiological and behavioral parameters (Hartenstein, 2017).

    DA neurons play a role in many aspects of insect brain function, which is not surprising since most parts of the brain are innervated by these neurons. Predictably, DA neurons projecting towards the mushroom body will be involved in learning and memory, whereas DA neurons associated with other compartments will have other functions. It is of course possible that intricate, not yet resolved interactions take place between the different DA neurons. One fundamental role of DA neurons appears to be to modulate overall motor activity (endogenous arousal; sleep vs wakefulness), as well as the responsiveness of brain neurons to specific sensory stimuli (Hartenstein, 2017).

    Three clusters of DA neurons of the adult brain, PPL1, PPL2, and PAM1, innervate the vertical lobe/spur (also called 'heel'), the calyx, and the medial lobe of the mushroom body (MB), respectively. Experimental studies link each of these clusters to memory formation. PPL1 neurons were identified as part of the circuit required for aversive odor memory, but (in part) also play a role in reward learning. Thus, several PPL1 neurons carry information reflecting energy build up during the formation of long term memory (Musso, 2015). In the larva, the DL1 (=PPL1 in adult), but also other DA groups (DM1, DM2) sense amino acid imbalances and control food intake (Bjordal, 2014). PAM1 neurons, innervating different domains of the medial lobes of the MB, also form part of the pathway that carries both aversive (electric shock) and reward stimuli (e.g., sweet taste; water) towards the MB. Different subsets of PAM1 neurons appear to be involved in the formation of short term and long term memories (Yamagata, 2015). Aggressive behavior is modulated by DA neurons of the PPM3 group whose neurons widely innervate the central complex. Members of the PPM3 cluster of DA neurons also control arousal and ethanol-induced locomotion (Hartenstein, 2017).

    An important step forward in understanding of DA neuronal function would be to decipher how DA synapses, as well as different DA receptors, are integrated into the microcircuitry of the brain. Progress towards this has been made in the mammalian prefrontal cortex, where DA axons innervate both projection neurons (glutamatergic, excitatory pyramidal cells) as well as local interneurons (inhibitory GABAergic cells). Pyramidal neurons form direct, reciprocal excitatory connections; in addition, local different types of interneurons form recurrent inhibitory and disinhibitory loops. The electrical activity of this structured network depends on the balance between excitatory and inhibitory elements; for example, elevated excitatory synapse activity, or depressed inhibitory synapse activity, would result in an abnormal spread of activation throughout the prefrontal cortex. Dopamine plays a key role in modulating this balance. The exact pattern of dopamine receptor expression in pyramidal neurons and inhibitory interneurons, and of the distribution of DA neurons within the microcircuitry of the prefrontal cortex, specifies the role of the DA system (Hartenstein, 2017).

    The relationship between DA terminals, DA receptors, projection neurons, and local interneurons has not been worked out in the Drosophila brain. Thus, it is not clear for the mushroom body, central complex, or any other compartment innervated by DA neurons what type of neuron is the direct target of the DA input. Attempts to reconstruct neuronal connectivity, in the larval brain, from serial section electron microscopy will hopefully provide answers to these questions. Thus, given their characteristic projection, branching pattern, and possibly ultrastructural characteristics (such as size and shape of transmitter vesicles), it should be possible to identify DA neurons in the serial EM stacks, and reconstruct their exact position within the neuronal circuits of different brain compartments. The association of DA neurons with specific lineages plays an important role within this undertaking. Thus, trajectories of primary lineages, including those containing the DA neurons, have been mapped for the early larval brain. As a result, DA lineages can be identified and their connectivity was abolished with the help of a serial EM stack (Hartenstein, 2017).

    Dopamine and serotonin are both required for mate-copying in Drosophila melanogaster

    Mate-copying is a form of social learning in which the mate-choice decision of an individual (often a female) is influenced by the mate-choice of conspecifics. Drosophila melanogaster females are known to perform such social learning, and in particular, to mate-copy after a single observation of one conspecific female mating with a male of one phenotype, while the other male phenotype is rejected. This study shows that this form of social learning is dependent on serotonin and dopamine. Using a pharmacological approach, dopamine or serotonin synthesis in adult virgin females with 3-iodotyrosine (3-IY) and DL-para-chlorophenylalanine (PCPA), respectively, and then their mate-copying performance was tested. While control females without drug treatment copied the choice of the demonstrator, drug-treated females with reduced dopamine or serotonin chose randomly. To ensure the specificity of the drugs, the direct precursors of the neurotransmitters, either the dopamine precursor L-3,4-dihydroxyphenylalanine (L-DOPA) or the serotonin precursor 5-L-hydroxytryptophan (5-HTP) were given together with the drug, (respectively 3-IY and PCPA) resulting in a full rescue of the mate-copying defects. This indicates that dopamine and serotonin are both required for mate-copying. These results give a first insight into the mechanistic pathway underlying this form of social learning in D. melanogaster (Monier, 2018).

    Aminergic signaling controls ovarian dormancy in Drosophila

    In response to adverse environmental conditions many organisms from nematodes to mammals deploy a dormancy strategy, causing states of developmental or reproductive arrest that enhance somatic maintenance and survival ability at the expense of growth or reproduction. Dormancy regulation has been studied in C. elegans and in several insects, but how neurosensory mechanisms act to relay environmental cues to the endocrine system in order to induce dormancy remains unclear. This fundamental question was examined in Drosophila by genetically manipulating aminergic neurotransmitter signaling in Drosophila melanogaster. Both serotonin and dopamine were found enhance adult ovarian dormancy, while the downregulation of their respective signaling pathways in endocrine cells or tissues (insulin producing cells, fat body, corpus allatum) reduces dormancy. In contrast, octopamine signaling antagonizes dormancy. These findings enhance understanding of the ability of organisms to cope with unfavorable environments and illuminate some of the relevant signaling pathways (Andreatta, 2018).

    A population of interneurons signals changes in the basal concentration of serotonin and mediates gain control in the Drosophila antennal lobe

    Serotonin (5-HT) represents a quintessential neuromodulator, having been identified in nearly all animal species where it functions in cognition, motor control, and sensory processing. In the olfactory circuits of flies and mice, serotonin indirectly inhibits odor responses in olfactory receptor neurons (ORNs) via GABAergic local interneurons (LN)s. However, the effects of 5-HT in olfaction are likely complicated, because multiple receptor subtypes are distributed throughout the olfactory bulb (OB) and antennal lobe (AL), the first layers of olfactory neuropil in mammals and insects, respectively. For example, serotonin has a non-monotonic effect on odor responses in Drosophila projection neurons (PNs), where low concentrations suppress odor-evoked activity and higher concentrations boost PN responses. Serotonin reaches the AL via the diffusion of paracrine 5-HT through the fly hemolymph and by activation of the contralaterally projecting serotonin-immunoreactive deuterocerebral interneurons (CSDns): the only serotonergic cells that innervate the AL. Concentration-dependent effects could arise by either the expression of multiple 5-HT receptors (5-HTRs) on the same cells or by populations of neurons dedicated to detecting serotonin at different concentrations. This study identified a population of LNs that express 5-HT7Rs exclusively to detect basal concentrations of 5-HT. These LNs inhibit PNs via GABAB receptors and mediate subtractive gain control. LNs expressing 5-HT7Rs are broadly tuned to odors and target every glomerulus in the antennal lobe. These results demonstrate that serotonergic modulation at low concentrations targets a specific population of LNs to globally downregulate PN odor responses in the AL (Suzuki, 2020).

    Serotonin (5-HT) represents a quintessential neuromodulator, having been identified in nearly all animal species where it functions in cognition, motor control, and sensory processing. In the olfactory circuits of flies and mice, serotonin indirectly inhibits odor responses in olfactory receptor neurons (ORNs) via GABAergic local interneurons (LNs). However, the effects of 5-HT in olfaction are likely complicated, because multiple receptor subtypes are distributed throughout the olfactory bulb (OB) and antennal lobe (AL), the first layers of olfactory neuropil in mammals and insects, respectively. For example, serotonin has a non-monotonic effect on odor responses in Drosophila projection neurons (PNs), where low concentrations suppress odor-evoked activity and higher concentrations boost PN responses. Serotonin reaches the AL via the diffusion of paracrine 5-HT through the fly hemolymph and by activation of the contralaterally projecting serotonin-immunoreactive deuterocerebral interneurons (CSDns): the only serotonergic cells that innervate the AL. Concentration-dependent effects could arise by either the expression of multiple 5-HT receptors (5-HTRs) on the same cells or by populations of neurons dedicated to detecting serotonin at different concentrations. This study identified a population of LNs that express 5-HT7Rs exclusively to detect basal concentrations of 5-HT. These LNs inhibit PNs via GABAB receptors and mediate subtractive gain control. LNs expressing 5-HT7Rs are broadly tuned to odors and target every glomerulus in the antennal lobe. The results demonstrate that serotonergic modulation at low concentrations targets a specific population of LNs to globally downregulate PN odor responses in the AL (Suzuki, 2020).

    This study identified a subset of GABAergic LNs that are responsible for signaling low levels of 5-HT in the AL. The 5-HT7R is well suited for detecting changes in basal concentrations of 5-HT, as it is the highest affinity serotonin receptor in Drosophila. Interestingly, 5-HT7R-expressing LNs in the R70A09 promoter line do not appear to express any other 5-HTRs, thus suggesting they may be dedicated cellular sensors of low 5-HT levels. LNs are an ideal target for modulation as they influence several aspects of olfactory coding in both flies and mice. Serotonin is also a potent modulator of GABAergic transmission in vertebrates , and 5-HT targets local interneurons in the olfactory bulb (OB) as well. LNs expressing 5-HT7R are non-specific in their glomerular innervation patterns and odor responses, suggesting a general role for their modulation. Indeed, it was observed that manipulations of basal levels of serotonin signaling had similar effects across glomeruli encoding pheromones, private odors, and broadly activating public odors. However, it is critical to note 5-HT7R expression likely differs across glomeruli. This study demonstrated that DA1 PNs do not express 5-HT7R, but other PNs likely do. Multiple cell classes in the AL express 5-HT7R and may do so in a glomerulus-specific manner. Thus, it is likely the exact effect of 5-HT differs slightly across glomeruli and is dependent on the unique expression pattern of 5-HT7R across cell types within each glomerulus. Glomerular-specific effects of 5-HT have been reported previously. Importantly, data regarding expression patterns of 5-HT7R come from approaches that label neurons according to promoters for these receptors. Although it was possible to discern which cell classes express specific 5-HTRs, there are no data to verify whether individual LNs can traffic receptors to dendrites innervating specific glomeruli. Future studies employing physiology or novel molecular tools will be vital for determining how 5-HTRs are dispersed across the AL to modulate PNs innervating different glomeruli (Suzuki, 2020).

    The best characterized form of inhibition in the AL and OB is presynaptic inhibition that targets ORN terminals and mediates divisive gain control. Divisive gain control is important, as it prevents the saturation of downstream PN and mitral cell responses, allowing olfactory circuits to encode odors over a wider range of concentrations. This form of gain control also allows the postsynaptic cell to utilize its entire dynamic range as the strength of presynaptic input increases. Finally, inhibition targeting ORNs leaves the window of temporal integration in PNs unaltered by inhibitory conductances. Interestingly, the results suggest that low-concentration 5-HT may specifically shift the balance between pre- and post-synaptic inhibition and mediate subtractive gain control. The postsynaptic inhibition arises in part from a monosynaptic connection between 5-HT7R-expressing LNs and PNs. Such direct connections from LNs onto PNs are supported by electron microscopy (EM) reconstructions of the AL in both the adult fly and larvae. The properties of postsynaptic inhibition are distinct from those described above. Postsynaptic inhibition will suppress PN firing from a broader distribution of inputs, rather than just its cognate ORNs. Subtractive gain control reduces the maximum firing rate of a neuron, thus diminishing its influence on perception, regardless of stimulus strength. This may be advantageous for suppressing behavior when sensory stimuli are presented in the wrong context. The utilization of divisive and subtractive gain control within the same circuit is not unique to olfaction, as it has also been described in the attentional regulation of vision (Suzuki, 2020).

    An alternative function for the suppression by low-concentration 5-HT may be to provide stability to olfactory processing under modulation. Data from the lobster stomatogastric ganglion (STG) indicate that dopamine has opposing actions on the same neurons, depending on its concentration. This mechanism has a homeostatic effect and stabilizes the motor output. Similarly, this study found that low and high concentrations of serotonin also have opposing effects, which are likely mediated by different receptors on different cells. Bidirectional modulation by the same transmitter has been reported in numerous other systems, suggesting it may be a common feature of the central nervous system across diverse phylogenetic groups. Indeed, as in flies, odor responses in vertebrate mitral cells are also boosted by methysergide and exogenous 5-HT. This suggests that bidirectional modulation by 5-HT may be a universal theme in olfaction (Suzuki, 2020). a

    Neurochemical organization of the Drosophila brain visualized by endogenously tagged neurotransmitter receptors

    Neurotransmitters often have multiple receptors that induce distinct responses in receiving cells. Expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits. This study describes a comprehensive library of reporter strains in which a convertible T2A-GAL4 cassette is inserted into endogenous neurotransmitter receptor genes of Drosophila. Using this library, the expression of 75 neurotransmitter receptors was profiled in the brain. Cluster analysis reveals neurochemical segmentation of the brain, distinguishing higher brain centers from the rest. By recombinase-mediated cassette exchange, T2A-GAL4 was converted into split-GFP and Tango to visualize subcellular localization and activation of dopamine receptors in specific cell types. This reveals striking differences in their subcellular localization, which may underlie the distinct cellular responses to dopamine in different behavioral contexts. These resources thus provide a versatile toolkit for dissecting the cellular organization and function of neurotransmitter systems in the fly brain (Kondo, 2020).

    The comprehensive collection of T2A-GAL4 lines generated in this study enabled performance of a brain-wide expression profiling of endogenous neurotransmitter receptors at high resolution. The T2A-GAL4 knockin system can faithfully recapitulate endogenous gene expression, without disrupting the function of the tagged protein in most cases (Kondo, 2020).

    The configuration of T2A-GAL4 knockin system has several key features that together make it advantageous to existing methods and resources. The first is the choice of the insertion site. C-terminal insertion used in this system is less likely to disturb the endogenous expression of the target than N-terminal or internal insertion, as it does not block communication between the promoter and intronic enhancers. Most neurotransmitter receptor genes contain multiple large introns, which are known to contain important cis-regulatory elements. Second, the inserted GAL4 transgene is followed by the endogenous 3' UTR of the target gene in this system. 3' UTR sequences are known to affect gene expression by regulating mRNA stability and subcellular localization. Importantly, recent studies in Drosophila have revealed that many neuronally expressed genes have long 3' UTR sequences that play critical roles in proper expression. Thus, the T2A-GAL4 knockin system recapitulates gene expression not only at the transcriptional level but also at the post-transcriptional level. Last, recombinase sites flanking the T2A-GAL4 cassette allow replacement of T2A-GAL4 with any other reporter genes by recombinase-mediated cassette exchange (RMCE). Unidirectional RMCE in this system offers a more rapid and straightforward way of transgene replacement than other systems. A series of exchange vectors was developed for converting T2A-GAL4 lines into other transcriptional activators, fluorescent fusions, and activity reporters. This versatility of this resource allowed the characterization not only expression patterns but also the subcellular localization of dopamine receptor proteins, as well as to visualize the dynamic regulation of receptor levels and activity (Kondo, 2020).

    Recently, a large-scale collection of gene-trap lines based on the MiMIC system and a T2A-GAL4 collection similar to the one described in this study have been developed. Both the resources and these collections represent versatile platforms for endogenous gene tagging. MiMIC insertions are located in coding introns and produce an internal GFP fusion. A previous study showed that ~30% of MiMIC-GFP insertions disrupt protein function and they could also disrupt subcellular localization. Although C-terminal tags are less likely to affect protein folding, they could disturb the function of certain proteins, in which the C terminus is modified or is important for interaction with other proteins. Thus, these collections complement each other in many respects, allowing users to choose reagents optimal for their studies (Kondo, 2020).

    Systematic clustering analysis of neuropils identified several brain regions with characteristic expression profiles of neurotransmitter receptors. Among others, neuropils constituting the MB and the central complex were conspicuously different from the rest of the brain. This observation is in line with their unique inter-neuropil connectivity previously revealed by connectome analysis: both the MB and the central complex lack overt connectivity with the peripheral nervous system and are considered higher order integrative centers of the brain. It is speculated that the unique combinations of receptors in these centers provide the basis of complex information processing in multi-modal integration (Kondo, 2020).

    At the level of individual cells, combinatorial expression of receptors could define the diverse responses of neurons to inputs from outside. Enormous complexity was observed in the expression profiles of transmitter receptors across the brain. The result of clustering analysis shows that receptors for the same ligand molecule or the same ligand type have similar expression patterns. Certain receptors expressed in the same neuron might function cooperatively by forming heterooligomers to recruit different G proteins or to form ion channels with different property. Specific functions of such receptor complexes will further contribute to the diversity of cellular responses, thereby augmenting the computational capacity of the neuronal circuit (Kondo, 2020).

    All dopamine receptors were expressed in the MB despite the fact that they have clearly distinct functions in fly behavior. This study confirmed co-expression of multiple dopamine receptors in individual Kenyon cells by simultaneous visualization of two receptors. This observation corroborates the results of a recent singe-cell transcriptome analysis in the fly brain. It is, however, in stark contrast to the situation in the mammalian striatum, where D1 and D2 receptors are expressed in segregated cell populations and mediate the bidirectional dopamine inputs. In the fly, segmentation was found of the neuronal cell membrane by differential subcellular localization of receptor proteins. Furthermore, various tagging approaches of endogenous receptors revealed unexpected subcellular localization of DopEcR as well as experience-dependent regulation of protein levels and activity. It is thus proposed that differential receptor localization along the cell membrane underlies distinct subcellular responses to dopamine input in different contexts (Kondo, 2020).

    Drosophila Ptp4E regulates vesicular packaging for monoamine-neuropeptide co-transmission

    Many neurons influence their targets through co-release of neuropeptides and small molecule transmitters. Neuropeptides are packaged into dense-core vesicles (DCVs) in the soma and then transported to synapses, while small molecule transmitters such as monoamines are packaged by vesicular transporters that function at synapses. These separate packaging mechanisms point to activity, by inducing co-release, as the sole scaler of co-transmission. Based on screening in Drosophila for increased presynaptic neuropeptides, the receptor protein tyrosine phosphatase (Rptp) Ptp4E was found to post-transcriptionally regulate neuropeptide content in single DCVs at octopamine synapses. This occurs without changing neuropeptide release efficiency, transport and DCV size measured by both STED super-resolution and transmission electron microscopy. Ptp4E also controls presynaptic abundance and activity of the vesicular monoamine transporter (VMAT), which packages monoamine transmitters for synaptic release. Thus, rather than rely on altering electrical activity, the Rptp regulates packaging underlying monoamine-neuropeptide co-transmission by controlling vesicular membrane transporter and luminal neuropeptide content (Tao, 2019).

    Synaptic complexity is enhanced by co-transmission with small molecules and bioactive peptides. The two transmitter classes differ in their postrelease distances traveled and durations of action, thus providing mechanisms for rapid point-to-point control and slow neuromodulation of circuits, development and behavior. Furthermore, from a cell biology perspective, transmission by small molecules and neuropeptides is distinguished by different vesicular loading mechanisms. The genetic results presented here are remarkable because (a) they reveal increased transmitter packaging, when past genetic screens have only yielded mutants that reduce vesicular packaging; (b) control of vesicular packaging varied between neuron subtypes based on differential Rptp expression, which represents a new mechanism for generating variation in co-transmission in the nervous system. Furthermore, this result is intriguing in the context of monoaminergic neurons because Ptp4E interacts genetically with α-synuclein toxicity in Drosophila. Given that synuclein is implicated in Parkinson's disease, DCV fusion pore dynamics and the early secretory pathway, the results here suggest that the mechanistic relationship between Rptps and synuclein may be broader than previously recognized; and (c) presynaptic abundance of a small-molecule vesicular membrane transporter and luminal neuropeptides are regulated in parallel. This shows that regulation of co-transmission is not limited to control of activity-induced vesicle exocytosis. Instead, an Rptp regulates vesicular packaging of both small-molecule and peptide neurotransmitters that underlies co-release (Tao, 2019).

    How can a single Rptp simultaneously modify vesicular loading of both monoamines and neuropeptides? Peptidergic neurotransmission relies on packaging of neuropeptides in the soma, where they condense in the TGN and are sorted into DCVs. There is little DCV circulation in octopamine terminals because of their extensive axonal arbors and numerous boutons. Therefore, Rptp regulation of neuropeptide content of individual DCVs likely originates prior to axonal transport. VMAT is also processed in the TGN to be sorted into DCVs and small synaptic vesicles, rather than proceeding through the constitutive secretory pathway. A recent study found that knockdown of the TGN protein HID-1 reduces DCV luminal cargo and VMAT in DCVs by affecting sorting and DCV production. The coordinated effects on neuropeptides and VMAT are reminiscent of the results presented in this study, but the Ptp4E effect on packaging was not associated with a change in DCV number or transport. Therefore, the uncoupling of DCV number from packaging is indicative of a novel cell biological mechanism. With this in mind, a possible explanation for the effect of inhibiting Ptp4E is that the tyrosine phosphorylation stimulates TGN sorting of luminal and vesicle membrane content without changing DCV number or size. By this mechanism, Rptp regulation of vesicular packaging in the soma could scale co-release at the distal synaptic ending (Tao, 2019).

    These results pose the question of the site of Ptp4E function. Rptps often mediate signaling triggered by cell-cell contacts. For example, the presynaptic Rptp Lar is activated by muscle Syndecan during development of the NMJ. By analogy, it is possible that presynaptic Ptp4E governs retrograde signaling (e.g., by interrupting tyrosine kinase-dependent mechanisms). In favor of this hypothesis, the closely related Rptp Ptp10D is found on axons in the embryo, where it is positioned to regulate axonal guidance during development. However, the potential involvement of vesicle biogenesis and the unknown localization of Ptp4E raise the possibility that somatic Ptp4E is responsible for synaptic effects. Novel tools to differentially control of Rptp activity by compartment (i.e. soma versus terminal) will be needed to distinguish between these possibilities. Regardless of the cellular location of Ptp4E signaling, the mechanism discovered here (i.e. coincident control of packaging of neuropeptides and small-molecule transmitters) represents a previously unknown cell biological strategy for regulating synaptic co-transmission (Tao, 2019).

    Previous experiments have shown that increased VMAT leads to enlargement of vesicles and greater vesicular monoamine storage, but this effect was not seen in this study. Notably, the mechanistic basis of the VMAT expression effect on vesicle size is not understood because thermodynamics with a simple system suggests that maximal vesicular monoamine concentration should be reached even with one VMAT per vesicle. Therefore, to explain the previously observed effect on vesicle size, some other factor, such as monoamine leakage or membrane flexibility, must come into play. It is suggested that these parameters might differ in the synaptic terminals examined in this study. Alternatively, in contrast to the spherical vesicles studied previously, the DCVs in octopamine neurons are ovoid. Therefore, the current analysis of largest dimension cannot exclude that the narrow axis of these DCVs increased. According to the latter scenario, increases in vesicular volume and membrane surface area could have been undetected with the methodology used in the current study (Tao, 2019).

    What would the expected consequences be of upregulating VMAT and neuropeptides in vesicles? Upregulating VMAT will increase the speed of vesicle loading when there is exocytosis-endocytosis cycling or kiss-and-run release. Hence, increased VMAT will affect release more when vesicle emptying by release is most marked. In Drosophila, the effect of increased VMAT on behavior has not been examined. However, the dopamine precursor L-DOPA increases vesicular monoamine content and ameliorates Parkinsonian symptoms in humans. Thus, by analogy, it is suggested that octopaminergic signaling would be boosted by increased vesicular octopamine packaging induced by synaptic VMAT upregulation. Of course, increased co-transmission by neuropeptides could further alter octopamine action. Therefore, it would be interesting to explore how Rptps in the brain affect octopamine-dependent fly behaviors such as feeding and egg laying (Tao, 2019).

    Bidirectional regulation of glial potassium buffering: glioprotection versus neuroprotection

    Glia modulate neuronal excitability and seizure sensitivity by maintaining potassium and water homeostasis. A salt inducible kinase 3 (SIK3)-regulated gene expression program controls the glial capacity to buffer K(+) and water in Drosophila, however upstream regulatory mechanisms are unknown. This study identified an octopaminergic circuit linking neuronal activity to glial ion and water buffering. Under basal conditions, octopamine functions through the inhibitory octopaminergic G-protein-coupled receptor (GPCR) OctβR to upregulate glial buffering capacity, while under pathological K(+) stress, octopamine signals through the stimulatory octopaminergic GPCR OAMB1 to downregulate the glial buffering program. Failure to downregulate this program leads to intracellular glia swelling and stress signaling, suggesting that turning down this pathway is glioprotective. In the eag shaker Drosophila seizure model, the SIK3-mediated buffering pathway is inactivated. Reactivation of the glial buffering program dramatically suppresses neuronal hyperactivity, seizures, and shortened life span in this mutant. These findings highlight the therapeutic potential of a glial-centric therapeutic strategy for diseases of hyperexcitability (Li, 2021).

    K+ homeostasis in the nervous system is required to maintain a healthy level of neuronal activity. Neurons release K+ ions as they repolarize during action potentials. If this K+ builds up in the extracellular space, it can disrupt neuronal firing and set the stage for neuronal hyperexcitability. Conventional treatments for conditions of hyperexcitability mostly target neuronal channels. However, more than one-third of epilepsy patients suffer from medically intractable seizures, while others experience debilitating side effects as neuron-targeting treatments often interfere with healthy neuronal functions. Hence, there is an urgent need for innovative therapeutic approaches that improve seizure control. This study explored glial mechanisms that regulate K+ balance to modulate neuronal excitability and seizure sensitivity (Li, 2021).

    Glia buffer K+ stress by taking in excess K+ ions and osmotically obliged water molecules from the extracellular space. This function allows glia to control the extracellular level of K+ and thereby modulate neuronal excitability, raising the hope that glia may be targeted to restore K+ homeostasis and suppress hyperexcitability in epilepsy and stroke. Glial-centric therapeutic strategies for these conditions have not been possible, largely due to a lack of understanding of mechanisms controlling the glial K+ buffering capacity. Previous work discovered a signal transduction pathway that controls the glial capacity to buffer K+ and water in Drosophila (Li, 2019). SIK3 (salt inducible kinase 3) is an AMPK family kinase that sequesters histone deacetylase 4 (HDAC4) in the cytoplasm, thereby promoting myocyte enhancer-factor 2 (Mef2)-dependent expression of K+ and water transport molecules, including the Drosophila orthologs of aquaporin-4 (Drip) and SPAK (Frayed) a kinase necessary for activating the Na+/K+ transporter NKCC1 Ncc69. This glial program suppresses extracellular nerve edema and prevents neuronal hyperexcitability and seizure in Drosophila, suggesting that it plays an important role in glial maintenance of K+ and water homeostasis and is required for a healthy level of neuronal activity. This glial program is best characterized in wrapping glia, which ensheathe axons in the Drosophila peripheral nervous system (PNS) and are akin to vertebrate non-myelinating Schwann cells, and so are well positioned to regulate the ionic composition around PNS axons (Li, 2021).

    While this study has elucidated downstream effectors by which SIK3 regulates K+ and water buffering, the upstream signals that work through the SIK3 pathway to control glial buffering capacity are unknown. In other contexts, SIK3 integrates signals from different signal transduction pathways, coupling extracellular signals to changes in cellular responses (Choi, 2015; Wang, 2011; Wein, 2018). One of these pathways is G-protein-coupled receptor (GPCR)-cyclic AMP (cAMP)-protein kinase A (PKA) signaling, in which PKA directly inhibits SIK3 activity (Wang, 2011). A better understanding of upstream signaling mechanisms that regulate the SIK3 pathway and extracellular cues that modulate glial K+ buffering capacity will inform approaches to leverage this glial function for therapeutic benefits in diseases of hyperexcitability (Li, 2021).

    This study identified an octopaminergic circuit linking neuronal activity to glial K+ and water buffering. Octopamine regulates the glial buffering program via GPCR-dependent regulation of PKA, which inhibits SIK3. At baseline, octopamine acts through the inhibitory octopaminergic GPCR OctβR to activate the glial SIK3 pathway to promote K+ and water buffering and healthy levels of excitability. Under pathological K+ stress, in contrast, octopamine functions through the stimulatory octopaminergic GPCR OAMB1 to inhibit SIK3 and downregulate the glial capacity to buffer K+ and water. While loss of the SIK3 pathway leads to extracellular edema, constitutive activation of the pathway results in intracellular glial swelling and activation of stress signaling, suggesting that turning down this pathway protects glia. In eag shaker, a classic Drosophila hyperexcitable mutant with defective K+ channels, the SIK3-mediated glial buffering program is turned off, likely as a self-protective mechanism for glia. However, reactivation of this glial K+ and water buffering program dramatically suppresses neuronal hyperexcitability, seizures, and shortened life span in eag shaker. Therefore, the maintenance of a robust glial K+ and water buffering program in response to extreme neuronal excitability is beneficial to the organism despite the risks posed to glial health. Taken together, this study identifies a neuromodulatory circuit that links neuronal activity to glial K+ buffering and highlights the promise of a glial-centric therapeutic strategy that restores K+ and water homeostasis for control of hyperexcitability in diseases such as epilepsy and stroke (LiH, 2021).

    K+ dysregulation drives edema and neuronal hyperexcitability and can lead to brain damage in patients with epilepsy and stroke. Glia buffer K+ stress by taking up excess K+ ions and water to maintain a healthy extracellular environment for neuronal excitability. Previous work identified SIK3 as the central node of a signal transduction pathway that controls the glial capacity to buffer K+ and water in Drosophila (Li, 2019). This study uncovered upstream regulatory mechanisms controlling the SIK3 pathway and demonstrated that activation of this glial program can dramatically ameliorate the pathological consequences of neuronal hyperexcitability. An octopaminergic circuit couples neuronal activity to glia buffering, exerting a dual effect on SIK3-mediated K+ buffering: low levels of octopamine enhance the glial K+ buffering capacity, while high levels of octopamine inhibit this buffering program, likely to protect glia against extreme K+ stress. Glial-specific inhibition of HDAC4, a central repressor of SIK3 signaling, results in a constitutively active glial buffering program that dramatically suppresses seizure and extends life span in a classic epilepsy model. Hence, augmenting glial K+ and water buffering holds promise as a therapeutic approach for the treatment of seizures (Li, 2021).

    The glial SIK3/HDAC4/Mef2 pathway regulates expression of both fray/SPAK, a kinase controlling ion transporter activity, and aquaporin 4, a water channel essential for volume regulation (Leiserson, 2011; Li, 2019; Papadopoulos, 2004). The original analysis of SIK3 LOF mutants demonstrated that glial SIK3 inhibits extracellular edema, presumably by promoting the uptake of ions and water into glial cells. This is an essential activity of glia, since accumulation of K+ in the extracellular space around axons leads to hyperexcitability and heightened seizure susceptibility. Since peripheral nerves must continuously fire, this raised the question of why expression of fray and aquaporin 4 would be regulated by SIK3, which is best understood to integrate opposing signals such as feeding and fasting to control the levels of downstream transcriptional targets. If ion and water buffering were so essential, then why is expression of essential buffering proteins not constitutive? The findings of this study suggest that the downregulating glial K+ buffering capacity is a protective response that prevents pathological glial swelling. Activity-dependent glial swelling is a commonly observed physiological phenomenon, but when taking in an excess load of ions and water, glia may swell to the point of cytolysis. This study has shown that glia with enhanced K+ buffering capacity undergo cell swelling that is exacerbated by extreme K+ stress. These glia also exhibit enhanced JNK pathway activity, which responds to a variety of stressors, including mechanical stress caused by cell stretching. JNK plays a critical role in the regulation of apoptosis, suggesting that pathological swelling that results from enhanced K+ buffering could promote cell death. Taken together, these findings demonstrate that overactivity of glial SIK3 activity leads to intracellular edema while underactivity of glial SIK3 signaling leads to extracellular edema. Hence, SIK3 balances the need for robust extracellular buffering with the health of the glial cell. It is suggested that the ability of the SIK3 pathway to integrate disparate upstream signals is important for maintaining this balance between an extracellular milieu conducive to neuronal firing with intracellular volume regulation that maintains glial health (Li, 2021).

    Norepinephrine is a stress hormone that can exert anticonvulsant effects in the mammalian nervous system. Moreover, norepinephrine regulates astrocyte transporter activity. In Drosophila, octopamine is structurally and functionally analogous to norepinephrine and regulates a variety of stress responses, including aggression, alertness, and starvation. Octopamine is also similar to norepinephrine in that it exerts dual effects on cellular responses by signaling through receptors with antagonistic functions. In this study, it is proposed that octopamine bidirectionally controls SIK3-mediated glial K+ buffering in response to changes in neuronal excitability (Li, 2021).

    Like its mammalian counterpart, octopamine is normally maintained at low levels and is released upon physiological stimuli to help the system mount a stress response. Octopamine release can be enhanced by a surge in the level of extracellular K+, suggesting that its concentration correlates with the level of K+ stress in the nervous system. This study proposes a model in which octopamine release links activity-dependent K+ stress to glial K+ buffering. This model is supported by a number of key findings. First, octopamine is required for SIK3 signaling and this regulation occurs through the inhibitory octopaminergic GPCR Octβ1R. Second, elevated levels of either octopamine or K+ each inhibit the glial SIK3 pathway, and both of these effects require the stimulatory octopaminergic GPCR OAMB. Hence, elevated K+ works through octopaminergic signaling to regulate the SIK3 pathway and glial buffering. Finally, the glial SIK3 pathway is inhibited in the hyperexcitable eag,Sh mutant. It is proposed that basal levels of octopamine promote SIK3-dependent glial K+ buffering to cope with physiological K+ stress and maintain a healthy level of neuronal activity. When neurons become hyperexcitable, as would occur under pathological conditions, levels of extracellular K+ rise and more octopamine is released. High levels of octopamine will inhibit SIK3 activity and turn down glial K+ buffering, likely to protect glia from pathological cell swelling (Li, 2021).

    This model posits that different levels of octopamine differentially activate Oct&beta1R and OAMB to exert these dual effects on glial K+ buffering. Octβ1R couples to the inhibitory Gαi protein, whereas OAMB can function through the excitatory Gαs protein . How might octopamine differentially signal through both Octβ1R and OAMB when they bind to the same ligand, reside in the same cell, and have antagonistic functions? Potentially, Octβ1R and OAMB could have different binding affinities for octopamine, with Octβ1R having a higher affinity such that Octβ1R is preferentially activated at low levels of octopamine. Inhibitory Gαi-coupled Octβ1R would decrease cAMP levels and inhibit PKA activity, thereby relieving the inhibition on SIK3 to upregulate glial K+ buffering capacity. Conversely, octopamine would bind to the lower affinity OAMB when octopamine levels are high, as would occur during hyperexcitability. High levels of octopamine signaling through the excitatory Gαs-coupled OAMB would enhance PKA activity and downregulate the glial capacity to buffer K+ stress. Hence, different binding affinities would allow Octβ1R and OAMB to be activated by different octopamine concentrations. A similar differential regulation via opposing octopaminergic receptors residing in the same cell is described at the larval NMJ, where octopamine signals through inhibitory Octβ1R and excitatory Octβ2R to bidirectionally control synaptic function (Li, 2021).

    This study has defined an octopaminergic circuit that links neuronal activity to glial K+ buffering to maintain K+ homeostasis and neuronal excitability. Neuromodulators such as octopamine are useful for integrating synaptic activity over time and space rather than signaling rapidly and locally like classical neurotransmitters. Moreover, altering the glial transcriptional response will be reasonably slow to change physiological function since such changes require transcription and translation. Hence, this system is likely tuned to respond to average neuronal activity rather than to transient changes in neuronal firing. This pathway may work in concert with additional mechanisms for rapid tuning of glial ion transporter function as described in mammalian astrocytes (Li, 2021).

    This model for octopaminergic regulation of the glial SIK3-dependent ionic buffering program leaves open a number of important questions. First, is the octopamine released locally or does release from the central octopaminergic neurons diffuse widely to set the glial tone for ionic buffering? Second, while this study showed that a glial octopamine receptor is necessary for pathway modulation by octopamine, it would also be interesting to test whether direct activation of octopaminergic neurons is sufficient to regulate the glial SIK3 pathway. Similarly, is direct inhibition of octopaminergic neurons capable of rescuing the whole animal phenotypes of the eag, Sh mutants? Third, while this study has focused on octopaminergic regulation, in other systems the SIK3 pathway is regulated by additional upstream signals including the kinase Lkb1 and the insulin receptor. Input from these additional pathways would allow for regulation of glial ion and water buffering in locations that may not be accessible for octopaminergic neuromodulation. Finally, this analysis focused on peripheral nerves and peripheral glia. In future studies it will be important to assess the role of this SIK3 buffering program in the cortex glia that buffer ions around neuronal cell bodies (Li, 2021).

    With hyperexcitability, waves of action potentials can lead to increases in extracellular K+ that, if not properly buffered, will lead to neuronal depolarization and further hyperexcitability. This study has explored the role of glial buffering in a paradigmatic Drosophila seizure mutant, eag,Sh, that exhibits dramatic neuronal hyperexcitability, seizure behaviors, and shortened life span. Although it would be expected that eag,Sh larvae have an increased need for glial buffering, this study found that the glial SIK3 signaling pathway is downregulated, likely as part of the glial protective response described above. Since downregulating the glial SIK3 pathway is sufficient to cause hyperexcitability and seizures, whether this inhibition of SIK3 could be exacerbating the eag,Sh phenotypes was tested. HDAC4 is the key repressor of SIK3-regulated glial K+ buffering, and so SIK3 signaling was activated by glial knockdown of HDAC4. Reactivation of the glial buffering program dramatically suppressed neuronal hyperexcitability, seizures, and shortened life span in eag,Sh. This result is quite remarkable, since the neurons are still deficient for two extremely important K+ channels. Nonetheless, the pathological consequences for the organism are almost wholly mitigated by enhanced glial buffering. This highlights the potential of glial-centric therapeutic strategies for diseases of hyperexcitability (Li, 2021).

    Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octbeta2R cascade

    Drosophila has been used as a model system to study mechanisms of sleep regulation. The first studies on sleep in Drosophila revealed that they periodically enter a quiescence state that meets a set of criteria for sleep. Drosophila sleep is monitored normally by a Drosophila activity monitoring system (DAMS) and is defined as immobility for 5 min or longer which is a sleep bout. Drosophila sleep mainly happens at night, while a period of siesta is in the mid-day. For example, total sleep time is around 380 min (male) and 250 min (female) during the day time, and 480 min (male) and 490 min (female) during the night time in w1118 (Zhao, 2021).

    In Drosophila, central complex structures, especially the ellipsoid body (EB) and fan-shaped body (FSB), are important for sleep homeostasis regulation. Activation of dorsal FSB neurons is sufficient to induce sleep. The dorsal FSB also integrates some sleep inhibiting signals. Both dorsal FSB and EB ring 2 are important in sleep homeostasis. Recently, the helicon cells were found to connect the dorsal FSB and EB Ring 2, indicating that these EB and FSB are connected (Zhao, 2021).

    Multiple studies indicate that the epigenetic mechanisms are involved in circadian regulation. However, a direct link between epigenetic regulation and sleep homeostasis is not yet established (Zhao, 2021).

    Octopamine (OA) in Drosophila is a counterpart of vertebrate noradrenaline. Previous studies in Drosophila showed that OA is a wake-promoting neurotransmitter and plays an important role in regulating both sleep amount and sleep homeostasis. The mutants of the OA synthesis pathway show an increased total sleep. Activation of OA signaling inhibits sleep homeostasis, while in OA synthesis pathway mutants, an enhanced sleep homeostasis is observed. Study of the neural circuit responsible for the sleep/wake effect of OA showed that octopaminergic ASM neuronsproject to the pars intercerebralis (PI), where OAMB (one of the OA receptors)-expressing insulin-like peptide (ILP)-secreting neurons act as downstream mediators of OA signaling. However, the effects of manipulating ASM neurons or ILP-secreting neurons are much weaker than those observed by manipulating all OA secreting neurons. Moreover, the effect of octopamine is not completely suppressed in the OAMB286 mutant, arguing that another receptor or circuit may participate in this process (Zhao, 2021).

    Eight OA receptors are identified to date: OAMB, Octβ1R, Octβ2R, Octβ3R, TAR1, TAR2, TAR3, and Octα2R. Although the expression pattern of OA is identified, the endogenous expression profile of these receptors is lacking. A previous study demonstrated that the mushroom body-expressed OAMB mediates the sleep:wake effect of OA. Recently, Octβ2R was shown to be important for the OA effect on endurance exercise adaptation. How the versatility of OA function is mediated by the diverse array of its receptors needs further study. Moreover, the upstream regulatory mechanisms of OA receptors are still unknown (Zhao, 2021).

    A previous study showed that Stuxnet (Stx) is important in mediating Polycomb (Pc) protein degradation in the proteasome (Du, 2016). Stx, which is an ubiquitin like protein, mediates Polycomb (Pc) protein degradation through binding to the proteasome with a UBL domain at its N terminus and to Polycomb through a Pc-binding domain. stx level changes result in a series of homeotic transformation phenotypes. Pc is an epigenetic regulator functioning in Polycomb Group (PcG) Complexes. Although it is reported that PcG component E(Z) is involved in circadian regulation, the role of stx in adult physiological process is unknown (Zhao, 2021).

    This study identified the role of the epigenetic regulator stx in sleep regulation. stx positively regulates Octβ2R through regulation of Polycomb in the EB of the adult fly brain. Further study demonstrated that the Stuxnet-Polycomb-Octβ2R cascade plays an important role in sleep regulation. In order to elucidate the role of this Stuxnet-Polycomb-Octβ2R cascade in sleep regulation, the role of various Octβ receptors was systematically identified in sleep regulation. Octβ2R was found to be one of the receptors that mediates OA function in sleep homeostasis. More interestingly, it was found that stx was OA-responsive depending on the Octβ1R. Based on these data, it is proposed that the Stuxnet-Polycomb-Octβ2R cascade provides a feedback mechanism for OA signals to the EB to regulate sleep homeostasis and sleep amount (Zhao, 2021).

    This study highlights the importance of epigenetic regulation on sleep. Although epigenetic regulation was intensively studied in adult pathological processes such as cancer, epigenetic factors have been far less studied in other physiological processes such as sleep. This study provides an example of the maintenance role of PcG complex in sleep regulation. Although the core PcG complex component Pc is ubiquitously expressed, its regulator stx is tissue specifically distributed, and this distribution may keep appropriate activity of Pc as well as the PcG complex in a tissue-specific manner. The factors regulating the tissue specificity of stx expression need to be further investigated (Zhao, 2021).

    A previous study found that mutation of Octβ2R does not have an obvious sleep phenotype. The current data were compared with the published Octβ2Rf05679 mutant data. Although Octβ2Rf05679 mutant was shown not significantly affected total sleep, this study found that the Octβ2Rf05679 has mild effect on sleep. Other Octβ2R mutants were tested, and it was found that the male flies from these mutations indeed have sleep phenotype (Zhao, 2021).

    Published studies have shown that the sleep phenotype of octopamine pathway mutants is different between video-based method and DAM-based method. For example, based on DAM data, the TβH mutant resulted in increased sleep per day, while the same mutant showed decreased sleep based on video data. This study used the video-based method to repeat the TβH mutant phenotype. The results showed that compared with the control flies, the TβH mutant got significantly less sleep. This result is consistent with the previously published data. Through close observation of TβH mutant and control flies, this study found that this mutant has much more frequent grooming behavior than the controls. The TβH mutant and control flies were video recorded for 10 min between ZT3.5 and ZT4.5. The results showed a statistically significant increase of the total number of grooming case. The difference between video-based method and DAM-based method is that these grooming behaviors can be detected in video-based methods, but not in DAM-based methods. Multiple studies have established a positive correlation between octopamine treatment and grooming behavior. Theoretically, TβH allele should result in a decrease in octopamine synthesis. The opposite phenotype may be caused by increased tyramine in TβH mutant or by other feedback regulation. The alleles for Octβ2R receptor used in this study show a similar grooming behavior as the control flies. The previously published octβ2R knockout allele should be a stronger one. The difference of sleep phenotypes between video-based and DAM-based methods may be due to the grooming behavior induced by the massive decrease of octopamine detection. Or other unrelated effects caused by the compensation effect previously reported. One hypothesis is that the significant change of grooming behavior probably masks the sleep behavior. The relationship between grooming and sleep needs to be further clarified. The detection of the sleep phenotype without significant changes in grooming phenotype may be a better strategy to get reliable sleep phenotype. If the increase of grooming in TβH mutant is a side effect caused by the increased tyramine, the identification of the phenotypes of octopamine treatment or collective phenotype of octopamine receptors may be more reliable ways to draw conclusions on the function of octopamine. Furthermore, whether grooming is epistatic to sleep is a problem worthy of further study (Zhao, 2021).

    Two aspects of sleep homeostasis need to be further studied. First, this study found that Octβ2R and stx colocalize in a subset of EB neurons. In a previously study, EB R2 neurons were found to be responsible for sleep homeostasis regulation. The relationship of these two groups of EB neurons needs further study. Second, the OA-treated Octβ2R mutant has more sleep recovery than the control. This indicates that OA induces more sleep recovery in the condition of Octβ2R downregulation. It seems that in this condition OA induces certain pathways to counteract its role in sleep homeostasis. One possibility is that Octβ2R negatively regulates Octβ3R which results in increased sleep pressure in the absence of Octβ2R. Further studies are needed to clarify the mechanism (Zhao, 2021).

    The results suggest the stx-Pc-Octβ2R regulatory cascade serves as a buffering step for OA function in sleep homeostasis. Two-way regulation of OA on stx leads to reverse changes of stx-the more OA, the less stx and vice versa. Through the function of stx-Pc-Octβ2R regulatory cascade, the Octβ2R transcription is changed accordingly. Variation of Octβ2R transcription could buffer the OA response. As a result, the unfavorable effect of OA causing dramatic decrease of sleep amount and homeostasis could be compensated by its receptor (Zhao, 2021).

    Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation

    Environmental stressors induce changes in endocrine state, leading to energy re-allocation from reproduction to survival. Female Drosophila melanogaster respond to thermal and nutrient stressors by arresting egg production through elevation of the steroid hormone ecdysone. However, the mechanisms through which this reproductive arrest occurs are not well understood. This study reports that stress-induced elevation of ecdysone is accompanied by decreased levels of ecdysis triggering hormone (ETH). Depressed levels of circulating ETH lead to attenuated activity of its targets, including juvenile hormone-producing corpus allatum and, as described in this study for the first time, octopaminergic neurons of the oviduct. Elevation of steroid thereby results in arrested oogenesis, reduced octopaminergic input to the reproductive tract, and consequent suppression of ovulation. ETH mitigates heat or nutritional stress-induced attenuation of fecundity, which suggests that its deficiency is critical to reproductive adaptability. These findings indicate that, as a dual regulator of octopamine and juvenile hormone release, ETH provides a link between stress-induced elevation of ecdysone levels and consequent reduction in fecundity (Meiselman, 2018).

    Evidence presented in this study establishes a new paradigm for Drosophila reproduction, wherein stressful conditions arrest egg production via a hormonal cascade involving reciprocal ecdysone and ETH signaling. As steroid levels fluctuate in response to stress, so too does ETH, a consequence of steroid-regulated changes in Inka cell secretory competence. ETH activates two downstream targets: the JH-producing corpus allatum and modulatory OA neurons innervating the ovary and oviducts. This study characterized the nature of ETH dependence, and assigned function and context to a newly recognized hormonal axis governing reproductive responses to stress (Meiselman, 2018).

    Previous report showed that ETH is an obligatory allatotropin, promoting oogenesis and fecundity through JH production; consequently, ETH deficiency results in low JH levels and arrested oogenesis (Meiselman, 2017). The present work demonstrates that ovulation of stage 14 oocytes depends upon ETH activation of OA neurons innervating the ovary and oviduct. A comprehensive explanation is offered for the change in distribution of vitellogenic oocytes reported in EcR mutants or under conditions of high or low ecdysone, depending on stress levels. ETH deficiency or ETHR knockdown results in accumulation of stage 14 oocytes in the ovary due to ovulation block, and a mechanistic link between altered endocrine state and ovulation is provided (Meiselman, 2018).

    ETH promotes ovulation through activation OA neurons to induce contractions in the ovary and relaxation of the oviducts. It is interesting that ETH triggers calcium dynamics in vitro on distal axonal projections, suggesting ETH-stimulated OA release results from direct action of ETH on axons and/or nerve terminals. While ovary contractions in response to ETH exposure occur in both virgin and mated females, this study chose virgin females for analysis due to higher spontaneous contractile activity in mated females. This is likely due to actions of ovulin after insemination, which stimulate outgrowth of octopaminergic neurons innervating the oviduct. In virgin females, concentration-dependent ETH actions on the ovary are in the range predicted for activation of ETHR-A receptors (Meiselman, 2018).

    Acting through OA neurons, ETH mobilizes calcium in the epithelium enveloping the ovary, initiating bursts of contractions in the peritoneal sheath at the base of the ovary associated with ovulation. Although bath-applied ETH and OA are both sufficient to induce calcium mobilization in the oviduct epithelium, they induce distinctive response patterns. OA causes a rapid, sustained calcium wave with a slowly waning plateau following the peak response. ETH actions occur with longer latency and induce oscillatory calcium dynamics, which could be a consequence of periodic synaptic reuptake of OA by nerve terminals. No changes in intensity were observed between treatments or at different doses, suggesting a possible threshold effect. It is also interesting to note that calcium waves spread through the epithelial layer, suggesting that the epithelium is a functional syncytium, which undoubtedly aids in coordination of relaxation (Meiselman, 2018).

    Injection of mated females with either ETH or OA induces ovulation in vivo, whereas injected virgin females respond much more weakly. In order for ovulation to occur, OA causes follicle rupture inside the ovaries, a process requiring one to several hours ex vivo. It is hypothesized that mated females are in the proper endocrine state for ovulation, and thus follicle rupture may already be in progress before application of ETH or OA. As follicle rupture is the critical first step for egg-laying, this limiting factor would explain the length of time (up to 60 min) elapsed after physiological levels of ETH/OA are reached for in vivo ovulation to occur, given that ovary contraction and oviduct relaxation occur within seconds (Meiselman, 2018).

    Agents previously implicated in oviduct contractions were also examined, including tyramine, glutamate, and proctolin. While the ineffectiveness of tyramine and glutamate is not surprising, the negative result with proctolin is at variance with prior literature. Examination of proctolin-induced contractions revealed that they are localized to the distal tip (germaria) of the ovaries. Moreover, proctolin does not stimulate ovulation in vitro. It appears that the role of proctolin in Drosophila ovaries is more limited than in the well-studied locust oviduct (Meiselman, 2018).

    This study has shown that elevated ecdysone levels in response to heat and nutritional stress are associated with a drop in circulating ETH levels. It was previously hypothesized that the Inka cell secretory competence model governing ecdysis signaling during developmental stages may persist into adulthood (Meiselman, 2017). The results presented in this study support this hypothesis (Meiselman, 2018).

    Both stress and ETH deficiency have similar consequences for reproduction, namely arrested oogenesis and reduced ovulation, resulting in increased stage 14 egg retention and lower egg production. Progression of mid-oogenetic oocytes is directly correlated with JH levels, while OA release from reproductive tract neurons is necessary for ovulation. This study shows that arrested oogenesis and ovulation contributing to the ovariole profile observed in heat-stressed flies can be explained by ETH deficiency, which has a dual role in regulating JH levels and activity of OA neurons innervating ovaries and oviducts. Indeed, arrest of both oogenesis and ovulation deficiencies can be rescued by ETH, either through TRPA1 activation of Inka cells or direct injection of ETH1 (Meiselman, 2018).

    The mechanism through which elevated ecdysone leads to ETH deficiency was examined by performing rescue experiments designed to (1) suppress steroid signaling in Inka cells and (2) express the transcription factor βFTZ-F1, which confers secretory competence of Inka cells and is suppressed by high ecdysone levels. Although somewhat variable in their effectiveness, these manipulations resulted in clear rescue of oogenesis and ovulation in heat-stressed females, confirming that the thermal stress response operates through the influence of ecdysone on Inka cell secretion (Meiselman, 2018).

    Methoprene treatment increases progression of oogenesis but does not increase oviposition in stressed animals. In fact, this study observed a significant increase in eggs retained after methoprene treatment, suggesting that synthesis of mature eggs resumes with JH treatment, but ovulation remains impaired under conditions of elevated ecdysone and ETH deficiency. This suggests that ovulation provides a gating mechanism under stressful conditions, limiting egg production while conditions are suboptimal. A recent report suggested that normal ecdysone levels stimulate follicle rupture and ovulation, but that elevated levels inhibit follicle rupture (Knapp, 2017). The present work provides an additional mechanism for suppression of ovulation associated with elevated ecdysone levels: repression of ETH release leading to reduced OA neuron activity (Meiselman, 2018).

    It is interesting to note that wet starvation reduces ecdysone levels and increases ETH levels, whereas sugar starvation increases ecdysone levels and, as is shown in this study, increases ETH levels. Wet-starved females were precisely synchronized in mating on day 4, and began starvation (no nutrient source, wet KimWipe) 24 h later for an additional 24 h. mino acid-deprived females were group-raised until day 3, and groups were placed on agar + 10% sucrose for 24 h. Mating was not controlled in sugar-starved females, though it is known to influence ecdysone levels dramatically in the short term. Arguably the most interesting result is that ecdysone decrease led to elevated circulating ETH. This adds credence to the hypothesis that ETH and ecdysone levels are generally inversely correlated (Meiselman, 2018).

    Unique stresses may garner different endocrine responses because different types of cues require differential behavioral adaptation. The ability of a hormone to coordinate a wide variety of target tissues to change in state makes it a perfect tool for stress adaptation. As an organism encounters a new type of stress, they may adapt a new endocrine state to coordinate a tissue-wide response. Many hormones in closely related insects play markedly different roles, which evolve as rapidly as behavioral niches, but an endocrine core in E-ETH-JH is highly conserved, similar to the hypothalamic-pituitary-gonadal (HPG) axis among vertebrates. A hormonal network with competence to adjust reproductive output in response to environmental changes is undoubtedly a common phenomenon among multicellular organisms. The discovery of a stress response hormonal axis and, more aptly, a peptide hormone with the potential to alleviate stress-induced deficits in reproduction could be of particular relevance to the honey bee Apis mellifera. In recent years, Apis reproductives have been producing fewer progeny due to a variety of stressors, including temperature extrema. While proctolin has already been found to be a short-term reproductive stimulant in Apis queens, ETH is attractive as it can alter JH levels, which in turn may rescue poor pheromone production, the proximal cause of supersedure (Meiselman, 2018).

    Enabling cell-type-specific behavioral epigenetics in Drosophila: a modified high-yield INTACT method reveals the impact of social environment on the epigenetic landscape in dopaminergic neurons

    Epigenetic mechanisms play fundamental roles in brain function and behavior and stressors such as social isolation can alter animal behavior via epigenetic mechanisms. However, due to cellular heterogeneity, identifying cell-type-specific epigenetic changes in the brain is challenging. This study reports the first use of a modified isolation of nuclei tagged in specific cell type (INTACT) method in behavioral epigenetics of Drosophila melanogaster, a method called mini-INTACT. Using ChIP-seq on mini-INTACT purified dopaminergic nuclei, epigenetic signatures were identified in socially isolated and socially enriched Drosophila males. Social experience altered the epigenetic landscape in clusters of genes involved in transcription and neural function. Some of these alterations could be predicted by expression changes of four transcription factors (Hr38, sr, CrebA, and Cbt) and the prevalence of their binding sites in several clusters. These transcription factors were previously identified as activity-regulated genes, and their knockdown in dopaminergic neurons reduced the effects of social experience on sleep. This work work enables the use of Drosophila as a model for cell-type-specific behavioral epigenetics and establishes that social environment shifts the epigenetic landscape in dopaminergic neurons. Four activity-related transcription factors are required in dopaminergic neurons for the effects of social environment on sleep (Agrawal, 2019).

    gamma-Oryzanol produces an antidepressant-like effect in a chronic unpredictable mild stress model of depression in Drosophila melanogaster

    Chronic unpredictable mild stress (CUMS) is a valid model for inducing depression-like symptoms in animal models, causing predictive behavioral, neurochemical, and physiological responses to this condition. This work aims to evaluate the possible antidepressant effect of γ-oryzanol (ORY) in the CUMS-induced depressive model in male Drosophila melanogaster. The CUMS protocol was used to continue a previous study, mimicking a depressive state in these insects. Male flies were subjected to various stressors according to a 10-day randomized schedule and concomitantly treated with ORY or fluoxetine (FLX). After the experimental period, in vivo behavioral tests were performed (open field, forced swimming, aggressiveness test, mating test, male virility, sucrose preference index and light/dark test) and ex vivo analyses measuring serotonin (5HT). dopamine (DA). octopamine (OCT) levels and body weight. ORY-treated flies and concomitant exposure to CUMS did not exhibit obvious behaviors such as prolonged immobility or increased aggressive behavior, reduced male mating and virility behavior, and anxiolytic behavior, in contrast to ORY, not altering sucrose preference and body weight flies exposed to CUMS. ORY effectively prevented 5HT and OCT reduction and partially protected against DA reduction. The data are consistent and provide evidence for the use of ORY as a potential antidepressant compound (Araujo, 2020).

    Iron overload during the embryonic period develops hyperactive like behavior and dysregulation of biogenic amines in Drosophila melanogaster

    Iron (Fe) is used in various cellular functions, and a constant balance between its uptake, transport, storage, and use is necessary to maintain its homeostasis in the body. Changes in Fe metabolism with a consequent overload of this metal are related to neurological changes and cover a broad spectrum of diseases, mainly when these changes occur during the embryonic period. This work aimed to evaluate the effect of exposure to Fe overload during the embryonic period of Drosophila melanogaster. Progenitor flies (male and female) were exposed to ferrous sulfate (FeSO(4)) for ten days in concentrations of 0.5, 1, and 5 ​mM. After mating and oviposition, the progenitors were removed and the treatment bottles preserved, and the number of daily hatches and cumulative hatching of the first filial generation (F1) were counted. Subsequently, F1 flies (separated by sex) were subjected to behavioral tests such as negative geotaxis test, open field test, grooming, and aggression test. They have evaluated the levels of dopamine (DA), serotonin (5-HT), octopamine (OA), tryptophan and tyrosine hydroxylase (TH), acetylcholinesterase, reactive species, and the levels of Fe in the progenitor flies and F1. The Fe levels of F1 flies are directly proportional to what is incorporated during the period of embryonic development; a delay in hatching and a reduction in the number of the hatch of F1 flies exposed during the embryonic period to the 5mM Fe diet were observed, a fact that may be related to the reduction of the cell viability of the ovarian tissue of progenitor flies. The flies exposed to Fe (1 and 5 ​mM) showed an increase in locomotor activity (hyperactivity) and a significantly higher number of repetitive movements. In addition to a high number of aggressive encounters when compared to control flies. An increase was observed in the levels of biogenic amines DA and 5-HT and an increase in TH activity in flies exposed to Fe (1 and 5 ​mM) compared to the control group. It is concluded that the hyperactive-like behavior demonstrated in both sexes by F1 flies exposed to Fe may be associated with a dysregulation in the levels of DA and 5-HT since Fe is a cofactor of TH, which had its activity increased in this study. Therefore, more attention is needed during the embryonic development period for exposure to Fe overload (Poetini, 2021).

    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).

    A serotonin-modulated circuit controls sleep architecture to regulate cognitive function independent of total sleep in Drosophila

    Both the structure and the amount of sleep are important for brain function. Entry into deep, restorative stages of sleep is time dependent; short sleep bouts selectively eliminate these states. Fragmentation-induced cognitive dysfunction is a feature of many common human sleep pathologies. Whether sleep structure is normally regulated independent of the amount of sleep is unknown. This study shows that in Drosophila melanogaster, activation of a subset of serotonergic neurons fragments sleep without major changes in the total amount of sleep, dramatically reducing long episodes that may correspond to deep sleep states. Disruption of sleep structure results in learning deficits that can be rescued by pharmacologically or genetically consolidating sleep. Two reciprocally connected sets of ellipsoid body neurons were identified that form the heart of a serotonin-modulated circuit that controls sleep architecture. Taken together, these findings define a circuit essential for controlling the structure of sleep independent of its amount (Liu, 2019).

    This study describes a circuit that regulates of sleep structure without affecting the total amount of sleep. 5HT acts to enhance the response of 5HT7-GAL4+ neurons to basally active excitatory inputs. 5HT-dependent calcium signals are blocked by TTX, while its ability to increase cAMP is not, supporting the existence of these active excitatory inputs to 5HT7-GAL4+ cells. In contrast, VT-GAL4+ cells do not have basally active excitatory inputs. 5HT modulation of the circuit likely occurs primarily via inputs to 5HT7-GAL4+ neurons since the response of VT038828-GAL4 (VT-GAL4)+ neurons is weaker and lower affinity. Whether there are other, perhaps situationally active, inputs to this circuit is currently unknown (Liu, 2019).

    Within the ellipsoid body (EB) the circuit is complex. VT-GAL4+ neurons are functionally connected with the 5HT7-GAL4+ group. VT-GAL4+ neurons provide feedback inhibition to a subset of 5HT7-GAL4+ neurons, which enhances fragmentation, likely via output to non-central complex regions. How inhibition of a subset of the 5HT7-GAL4+ cells acts to modulate the behavioral output of the rest of the population is not yet clear, but it is noted that many of the 5HT7-GAL4+ cells are GABAergic. While all the details of the circuit's complex dynamics remain to be discovered, it is clear that this circuit has a profound and selective effect on sleep architecture (Liu, 2019).

    The circuit described in this study is modulated by 5HT, a neurochemical known to be important for regulation of behavioral states in many species. While 5HT in mammals is important in a wide variety of contexts, it was controversial for nearly half a century whether it promoted sleep or wakefulness. In Drosophila, 5HT has only been thought to promote sleep. The current data show that upregulation of serotonergic signaling can also induce sleep fragmentation, suggesting that 5HT's role in sleep in flies exhibits a complexity similar to that of its roles in mammals. The genesis of this apparently conserved complexity may be the extensive involvement of 5HT in non-sleep processes. For an animal in the wild, sleep has inherent risks: predation and loss of opportunities for mating or feeding are just a few. Sleep/wake systems in the brain must control arousal state in collaboration with systems that assess competing needs. 5HT, because it is central to so many critical behavioral circuits, is ideally poised to be an integration point for sleep and the general state of the animal. The diverse, circuit-specific, roles in sleep that 5HT exhibits across phyla may be a result of its ubiquity (Liu, 2019).

    The role this study has uncovered for 5HT as a regulator of sleep architecture aligns well with this idea. The daily neuronal activity profile reported by Tric-LUC, a calcium sensor that drives luciferase expression in response to neuronal activity, in sleep fragmentation-generating neurons maps to dawn and dusk, when crepuscular organisms such as fruit flies are most active. Fragmentation of sleep at these times would presumably be beneficial since flies would not enter into deep sleep states at times when they should be feeding and mating. Interestingly, the circuit described in this study accomplishes this feat by increasing P(doze), the probability of falling asleep from a wake state, leaving the scaling of P(wake), a parameter associated with dopamine and arousal, free to be modulated by other factors (e.g., danger from predation, appearance of potential mates). The fact that long sleep bouts can be prevented without putting the animal into a hyperaroused state is advantageous, allowing flexible responsiveness to changing conditions. The involvement of P(Doze), a parameter associated with sleep drive, is also congruent with the sleep-promoting role of 5HT in other brain regions (Liu, 2019).

    While controlled sleep fragmentation appears to assist in active period behavior, there is also a need for consolidated sleep. In both mammals and Drosophila, sleep has electrophysiologically distinct substrates with progressively higher arousal thresholds that appear in an ordered fashion during a sleep episode. The deeper sleep stages in mammals, REM and slow wave sleep, are strongly associated with maintenance of cognitive function. Fragmentation of sleep, because it truncates sleep episodes before deeper stages are reached, can result in a selective deprivation of deep sleep stages even when total sleep is not changed. In this study, it was demonstrated that decreasing sleep consolidation, without changing the amount of sleep, can disrupt associative learning. These results suggest that in Drosophila, like in mammals, there are time-dependent changes in the depth of sleep that are important for its beneficial effects. This idea is also supported by modeling and analysis of the structure of fly sleep, which indicate that there are time-dependent changes in the probability of sleep-wake transitions consistent with the existence of deep sleep stages that are only accessed in long sleep episodes (Liu, 2019).

    Fragmentation of sleep induced by activation of 5HT inputs to the EB also produced an increase in sleep after the activation was terminated. Excess sleep in the recovery day after a perturbation is a hallmark of a homeostatic process. Homeostatic regulation of total sleep time has been previously demonstrated in Drosophila, but the data suggest that there is also homeostatic regulation of sleep quality. In mammals, individual sleep substates have been demonstrated to be homeostatically regulated- selective deprivation of REM or slow wave sleep, in the absence of loss of total sleep time, drive rebound increases of the deprived stage and mechanical sleep fragmentation has been shown to lead to an increase in total sleep. The ability of the EB circuit in Drosophila to selectively modulate sleep structure, without changing the total amount of sleep, has allowed for the first time the selective probing of the cognitive importance of long sleep bouts and deep sleep stages in the fly. The fact that fragmentation triggers rebound sleep implies that these long sleep bouts may also be important for the general health benefits of sleep (Liu, 2019).

    Serotonergic modulation of visual neurons in Drosophila melanogaster

    Sensory systems rely on neuromodulators, such as serotonin, to provide flexibility for information processing as stimuli vary, such as light intensity throughout the day. Serotonergic neurons broadly innervate the optic ganglia of Drosophila. This study mapped of patterns of serotonin receptors in the visual system, focusing on a subset of cells with processes in the first optic ganglion, the lamina. Serotonin receptor expression was found in several types of columnar cells in the lamina including 5-HT2B in lamina monopolar cell L2, required for spatiotemporal luminance contrast, and both 5-HT1A and 5-HT1B in T1 cells, whose function is unknown. Subcellular mapping with GFP-tagged 5-HT2B and 5-HT1A constructs indicated that these receptors localize to layer M2 of the medulla, proximal to serotonergic boutons, suggesting that the medulla neuropil is the primary site of serotonergic regulation for these neurons. Exogenous serotonin increased basal intracellular calcium in L2 terminals in layer M2 and modestly decreased the duration of visually induced calcium transients in L2 neurons following repeated dark flashes, but otherwise did not alter the calcium transients. Flies without functional 5-HT2B failed to show an increase in basal calcium in response to serotonin. 5-HT2B mutants also failed to show a change in amplitude in their response to repeated light flashes but other calcium transient parameters were relatively unaffected. While serotonin receptor expression in L1 neurons was not detected, they, like L2, underwent serotonin-induced changes in basal calcium, presumably via interactions with other cells. These data demonstrate that serotonin modulates the physiology of interneurons involved in early visual processing in Drosophila (Sampson, 2020).

    A conserved role for sleep in supporting Spatial Learning in Drosophila

    Sleep loss and aging impair hippocampus-dependent Spatial Learning in mammalian systems. This study used the fly Drosophila melanogaster to investigate the relationship between sleep and Spatial Learning in healthy and impaired flies. The Spatial Learning assay is modeled after the Morris Water Maze. The assay uses a 'thermal maze' consisting of a 5X5 grid of Peltier plates maintained at 36-37°C and a visual panorama. The first trial begins when a single tile that is associated with a specific visual cue is cooled to 25°C. For subsequent trials, the cold tile is heated, the visual panorama is rotated and the flies must find the new cold-tile by remembering its association with the visual cue. Significant learning was observed with two different wild-type strains - Cs and 2U, validating the design. Sleep deprivation prior to training impaired Spatial Learning. Learning was also impaired in the classic learning mutant rutabaga (rut); enhancing sleep restored learning to rut mutants. Further, flies exhibited dramatic age-dependent cognitive decline in Spatial Learning starting at 20-24 days of age. These impairments could be reversed by enhancing sleep. Finally, Spatial Learning was found to requires dopaminergic signaling and that enhancing dopaminergic signaling in aged flies restored learning. These results are consistent with the impairments seen in rodents and humans. These results thus demonstrate a critical conserved role for sleep in supporting Spatial Learning, and suggest potential avenues for therapeutic intervention during aging (Melnattur, 2020).

    Serotoninergic Modulation of Phototactic Variability Underpins a Bet-Hedging Strategy in Drosophila melanogaster

    When organisms' environmental conditions vary unpredictably in time, it can be advantageous for individuals to hedge their phenotypic bets. It has been shown that a bet-hedging strategy possibly underlies the high inter-individual diversity of phototactic choice in Drosophila melanogaster. This study shows that fruit flies from a population living in a boreal and relatively unpredictable climate have more variable phototactic biases than fruit flies from a more stable tropical climate, consistent with bet-hedging theory. This study experimentally showed that phototactic variability of D. melanogaster is regulated by the neurotransmitter serotonin (5-HT), which acts as a suppressor of the variability of phototactic choices. When fed 5-HT precursor, boreal flies exhibited lower variability, and they were insensitive to 5-HT inhibitor. The opposite pattern was seen in the tropical flies. Thus, the reduction of 5-HT in fruit flies' brains may be the mechanistic basis of an adaptive bet-hedging strategy in a less predictable boreal climate (Krams, 2021).

    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).

    LAT1-like transporters regulate dopaminergic transmission and sleep in Drosophila

    Amino-acid transporters are involved in functions reportedly linked to the sleep/wake cycle: neurotransmitter synthesis and recycling, the regulation of synaptic strength, protein synthesis and energy metabolism. In addition, the existence of bidirectional relationships between extracellular content, transport systems and sleep/wake states is receiving emerging support. Nevertheless, the connection between amino-acid transport and sleep/wake regulation remains elusive. To address this question, this study used Drosophila melanogaster and investigated the role of LAT1 (Large neutral Amino-acid Transporter 1) transporters. This study shows that the two Drosophila LAT1-like transporters: JhI-21 and minidiscs (Mnd) are required in dopaminergic neurons for sleep/wake regulation. Down-regulating either gene in dopaminergic neurons resulted in higher daily sleep and longer sleep bout duration during the night, suggesting a defect in dopaminergic transmission. Since LAT1 transporters can mediate in mammals the uptake of L-DOPA, a precursor of dopamine, amino-acid transport efficiency was assessed by L-DOPA feeding. Downregulation of JhI-21, but not Mnd, reduced the sensitivity to L-DOPA as measured by sleep loss. JhI-21 downregulation also attenuated the sleep loss induced by continuous activation of dopaminergic neurons. Since LAT1 transporters are known to regulate TOR (Target Of Rapamycin) signaling, the role of this amino-acid sensing pathway in dopaminergic neurons was investigated. Consistently, it is reported that TOR activity in dopaminergic neurons modulates sleep/wake states. Altogether, this study provides evidence that LAT1 mediated amino-acid transport in dopaminergic neurons, is playing a significant role in sleep/wake regulation, and is providing several entry points to elucidate the role of nutrients such as amino-acids in sleep/wake regulation (Aboudhiaf, 2018).

    Emerging evidence suggests bidirectional relationships between extracellular space content and vigilance states, emphasizing the so far little explored sleep-regulatory role of the transmembrane transport of ions and small molecules. Sleep and wakefulness have a pervasive impact on brain cellular activities linked to neurotransmission, neuronal plasticity, neurotransmitter synthesis, nutrient supply, and waste elimination, relying on the efficient and precise coordination of transport systems. Investigating how transporters and underlying molecular and cellular mechanisms are involved in these mutual interactions requires the targeting of individual genes in specific cell types. This strategy is highly amenable to the Drosophila model. Among the large array of cellular transporters present in the genome and conserved between insects and mammals, the well-characterized large neutral amino acid transporters are particularly relevant given their role in neurotransmitter synthesis and recycling, in the regulation of synaptic strength, in protein synthesis and energy metabolism. In addition, the de novo synthesis of brain monoamines associated with wakefulness and neuromodulation, such as serotonin, dopamine, and noradrenaline, is dependent on large neutral essential amino acids provided by the blood (Aboudhiaf, 2018).

    The SLC7A5 (or large neutral amino-acid transporter, light chain or LAT1) and SLC7A8 (LAT2) amino acid transporters are present in most cell types and appear to play a prominent role in the Na+-independent transport of large branched and aromatic neutral amino acids. These transporters belong to the heterodimeric amino acid transporters (HAT) family and require co-expression of the CD98hc /4F2hc (SLC3A2) heavy chain, to which they can be covalently linked by a di-sulfur bridge. The heavy subunit does not appear to confer transport-specific properties, nor to be confined to HAT transporter function. In the mammalian brain, LAT1 is highly expressed in the cells of the blood-brain-barrier and is thought to play a critical role in providing the central nervous system with essential amino acids such as phenylalanine, tyrosine, leucine, and tryptophan, which are nutrients and precursors for monoamine synthesis. The uptake of leucine through LAT1 is a major activator signal for target of rapamycin complex 1 (TORC1), a cellular pathway dependent on the TOR kinase that controls protein synthesis, brain excitability, and plasticity. Reciprocally, inhibition of mTOR by rapamycin has been shown to significantly reduce the activity and the mRNA expression of LAT1. Despite a few pieces of evidence, it remains to be investigated whether LAT1, LAT2, or other SLC7A transporters are also localized in neurons, and whether their function is linked to sleep and wake. In Drosophila, the HAT family of transporters is represented by one heavy chain, CD98hc, and five light chains: juvenile hormone inducible-21 (JHI-21), minidiscs (MND), genderblind (GB), CG9413 protein, and CG1607 protein. The specific activity of these transporters cannot be easily predicted from their sequences and requires functional testing. JHI-21 and MND can transport leucine, are inhibited by BCH (2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and, at least for JHI-21, require CD98hc to become functional, thus classifying them as LAT1-LAT2 homologs. Both genes are expressed at high levels in the central nervous system and have been shown to play important neurophysiological functions. JHI-21 and GB regulate glutamatergic synaptic strength, primarily through the regulation of extracellular glutamate levels, and are, respectively, required in motor neurons and in glial cells. In a recent report, it has also been shown that MND is required to activate brain insulin-producing neurons in response to circulating leucine. Interestingly those same insulin-producing neurons are connected to wakefulness promoting circuits. This study used the molecular-genetic tools of Drosophila to investigate the potential impact of JhI-21 and Mnd downregulation in neuronal subsets on sleep/wake regulation (Aboudhiaf, 2018).

    Evidence is provided that the LAT-1 like transporters JhI-21 and Mnd are required in adult fly dopaminergic neurons to achieve adequate sleep/wake regulation. The results demonstrate that a downregulation of these transporters in dopaminergic neurons results in a decrease in wakefulness, under baseline conditions but also in conditions that increase dopaminergic transmission. This implies that the activity of dopaminergic neurons and/or their ability to release neurotransmitter requires JhI-21 and Mnd. These two amino acid transporters are the closest drosophila LAT1 homologs based on sequence and functional data, suggesting that some amino acid availability plays a critical role in dopaminergic neuronal function. Supporting this hypothesis, this study finds that downregulating the TOR pathway in dopaminergic neurons results in a decrease in wakefulness (Aboudhiaf, 2018).

    As in mammals, dopaminergic transmission plays a major role in Drosophila wakefulness and has been suggested to constitute a core ancestral regulator of arousal and sleep entry across invertebrates. Drosophila and mammals share homologs for genes playing a central role in dopamine synthesis, reuptake, and signaling. This includes two D1-like receptors and one D2-like dopamine receptor. Among those, the D1-like receptor Dop1R1 (dDA1) plays a prominent role in sleep/wake regulation. Dop1R1 mutant flies display high daily sleep, longer sleep bout duration, and normal waking activity, a phenotype that closely resembles the one obtained in this studu by downregulating JhI-21 or Mnd in dopaminergic neurons. The effect of Dop1R1 on sleep/wake depends on its expression in the dorsal fan-shaped body (dFB), a key sleep-wake regulatory structure, and on the release of dopamine by a very limited set of dopaminergic neurons projecting to the dFB and located in the PPL1 and PPM3 cluster. Thus, it is possible that Mnd and JhI-21 are required in those specific neurons to achieve normal sleep-wake regulation. Expressing Mnd and JhI-21 UAS-RNAi constructs in PPL1 and PPM3 neurons failed to produce an abnormal sleep/wake phenotype. This lack of effect may be attributable to the limited efficiency of the UAS-RNAi constructs. Alternatively, the inhibition of JhI-21 and Mnd may affect multiple dopamine dependent microcircuits throughout the brain and thus cannot be easily replicated by a more specific manipulation (Aboudhiaf, 2018).

    What function could JHI-21 and MND fulfill in dopaminergic neurons? At the glutamatergic neuromuscular junction, JHI-21 appears to regulate the clustering of post-synaptic glutamate receptors. JHI-21 does not appear to trigger directly the release of glutamate in this context, but possibly mediates the entry of amino acids such as leucine to activate molecular pathways controlling glutamatergic physiology. Although abnormal sleep/wake regulation was not observed when JhI-21 was inhibited in glutamatergic neurons, the results indicate that JhI-21 could play a role in dopaminergic physiology similar to the one hypothesized for the neuromuscular junction. The synthesis of brain monoamines depends on the supply of essential amino acids that are provided by food intake, thus requiring efficient cellular transport systems. Although dopaminergic neuron function is impaired when JhI-21 expression is downregulated, no evidence was found of reduced brain dopamine levels under baseline conditions. Dopamine levels were increased in the mutant flies fed with the dopamine precursor L-DOPA, an amino acid that has been shown to be transported by LAT1, further suggesting that JhI-21 expression is not critical for dopamine synthesis. In this experiment, dopamine synthesis could also take place ectopically in dopa-decarboxylase (ddc) expressing serotoninergic neurons, in which the UAS-JhI-21-RNAi construct was not expressed. Dopamine content was still increased, although moderately, in flies in which the UAS-JhI-21-RNAi construct was targeted to both dopaminergic and serotoninergic neurons using the ddc-Gal4 driver. The lack of impact of JhI21 downregulation on L-DOPA-induced increase in dopamine synthesis could be due to functional redundancy between JhI-21 and Mnd, or could be a consequence of the partial inhibition provided by the UAS-RNAi constructs. In contrast to those findings on tissue content, this study clearly observed that JhI-21 downregulation in dopaminergic neurons reduced the sensitivity to L-DOPA as measured with sleep loss, and significantly attenuated the sleep loss phenotype of the TH-GaL4 > UAS-TrpA1 hyperdopaminergic condition. This raises the possibility that JhI-21 mediates the entry of amino acids critical for the physiology of dopaminergic neurons, such as leucine that could activate TOR-dependent processes influencing protein synthesis and energy metabolism, but is not directly involved in dopamine synthesis through the transport of precursors such as tyrosine or phenylalanine. The phenotype observed in flies where both Rheb and JhI-21 are manipulated indicates that JhI-21 could be required for TOR pathway activation. This hypothetical model is conceivable since in mammals enriched intracellular leucine levels via LAT1 transport activate mTORC1. Alternatively, TOR signaling could modulate JHI-21 function and affect LAT1-like-dependent transport mechanisms required for neuronal activity. Elucidating the relationships between TOR signaling and LAT1-like transporters, and understanding how they play a critical role in dopaminergic neurons function will require further work (Aboudhiaf, 2018).

    This study showed that the MND and JHI-21 transporters are broadly expressed in the brain. However, targeting the JhI-21 and Mnd UAS-RNAi constructs to most nondopaminergic cell types, including the wake-promoting octopaminergic neurons, failed to affect sleep-wake regulation. This lack of effect may be attributable to the efficiency of genetic tools or alternatively to the more stringent requirement for these transporters in dopaminergic neurons. Further studies are warranted to further evaluate this question. Interestingly, accumulating pieces of evidence support the existence of a dynamic regulation for JhI-21 and Mnd: JhI-21 is modulated during larval development in close correlation with behavioral changes and in adult after long-term memory conditioning, whereas Mnd is differentially expressed after sleep deprivation. The expression of LAT1 transporters in mammalian dopaminergic neurons has not been investigated yet; however, a report using pharmacological methods suggested that such transporters could modulate neuronal activity. The fact that sleep deprivation induces changes in TOR signaling in the brain opens the possibility that LAT1 could be modulated by sleep/wake in mammals. Of note, sleep deprivation could induce changes in LAT1 expression at the blood-brain barrier (Aboudhiaf, 2018).

    In conclusion, this study reveals the role of LAT1-like transporters in the function of dopaminergic neurons, adding one more element to the array of cellular and molecular events affecting sleep/wake regulation. Since these transporters are known to be dynamically regulated by physiological cellular states, they provide an entry point to elucidate the role of nutrient in sleep/wake regulation (Aboudhiaf, 2018).

    Dopamine signaling in wake promoting clock neurons is not required for the normal regulation of sleep in Drosophila

    Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake- and sleep-promoting cell types. The large and small ventrolateral neurons (l-LN(v)s and s-LN(v)s) have been identified as wake-promoting neurons within the clock neuron network. The l-LN(v)s are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LN(v)s and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. This study tested this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. Dopamine application indeed increases cAMP levels and depolarizes the l-LN(v)s, but surprisingly, it does not result in increased firing rates. Down-regulation of the excitatory dopamine receptor, Dop1R1 in the l- and s-LN(v)s, but not of Dop1R2, abolished the depolarization of l-LN(v)s in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LN(v)s. Down-regulation of Dop1R1 or Dop1R2 receptors in the l- and s-LN(v)s does not affect sleep in males. Unexpectedly, a moderate decrease of daytime sleep was found with down-regulation of Dop1R1 and of nighttime sleep with down-regulation of Dop1R2. Since the l-LN(v)s do not utilize Dop1R2 receptors and the s-LN(v)s also respond to dopamine, it is concluded that the s-LN(v)s are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LN(v)s is not required for daytime arousal, but likely promotes nighttime sleep via the s-LN(v)s (Fernandez-Chiappe, 2020).

    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).

    Olfactory attraction to ethanol is regulated by two sets of serotonergic neurons. Two neurons -- the serotonergic CSD neurons -- counteract the inhibitory function of a second set of four serotonergic neurons. The enhancement of sensory input by altered serotonin signaling of the CSD neurons is stronger than more internal information that might influence the behavioral outcome. The observation that strong sensory information overrules internal conditions unravels a hierarchy of information that governs behavioral output (Xu, 2016).

    Prolonged serotonin signaling in approximately 83% of the serotonergic neurons using the Trh-Gal4 driver results in normal degree of attraction to 5% EtOH containing food odors. At the first glance, these finding suggest that dSert in these broad set of serotonergic neurons is not involved in the regulation of ethanol attraction. This apparently normal behavior is shared by other Sert knock out animals. For example Sert knock out mice develop normal conditioned place preference -- a model for the cocaine reward -- despite the fact that cocaine binds to Sert. However, breaking down the serotonergic networks underlying the regulation of the attraction to ethanol revealed that a dysregulation of serotonergic neurons can phenocopy normal behavior and that the identified serotonergic neurons under normal circumstance indeed regulate the olfactory attraction. The results show that compensation can occur on network level. The serotonergic neurons underlying the gating of olfactory attraction to ethanol innervate distinct brain regions normally involved in the regulation of olfactory responses. The attraction promoting serotonergic CSD neuron receives synaptic input from the ipsilateral antennal lobes from a broad set of glomeruli, collecting and exchanging olfactory information with the mushroom bodies and lateral horn and releasing information to the contralateral antennal lobe regions (Xu, 2016).

    The second set of inhibitory serotonergic neurons includes one neuron each from the IP and LP1 clusters that suppresses innate attraction to ethanol odor. The neuron of the IP cluster projects into the superior medial protocerebrum (smpr), posterior lateral protocerebrum (plpr), and the optic system. The smpr receives olfactory output both directly from the antenna lobes and indirectly from lobes of the LH. The LP1 neuron innervates the ventral lateral protocerebrum (vlpr), a region implicated as a multimodal integration center of sensory stimuli including olfactory information. This multimodal nature of this brain region is further supported by recent finding that a pair of similar serotonergic neurons is involved in aggression and mediating the hunger state of the fly. The fact that the alteration of serotonin signaling in the CSD neuron bypasses the inhibitory effect of the Sert3-Gal4-dependent neurons further supports that the inhibitory effect of those neurons is not due to loss of olfactory perception. Combined with the observation that activation of the Sert3-Gal4-dependent neurons alone does not cause attraction or aversion, these results show that these neurons regulate the execution of a function rather than being involved in the initial step of odor information processing or execution of the approach behavior. This is consistent with a role for prolonged serotonin signaling in response inhibition. This suppression of innate olfactory attraction to ethanol can be overruled by strengthening the constant sensory input to the olfactory system. The consequence of the failing inhibitory mechanism is that the likelihood of exposure to food odors containing ethanol increases, and the opportunity to ingest ethanol might increase in turn (Xu, 2016).

    The observation that strong sensory information overrules internal conditions also suggests that under normal circumstances both mechanisms require a tight temporal control. That timing matters is further substantiated by the observations that short term systemic application of a serotonin precursor and short term activation of the Sert3-Gal4 dependent subset of serotonergic neurons using opto-genetics reduce ethanol attraction, but long term intervention of serotonin signaling by dSertDN expression under the control of the Trh-Gal4 driver did not interfere with ethanol attraction. However the regulation of innate ethanol odor attraction is even more complex since other neurotransmitter systems are also involved in regulating the approach to an olfactory stimulus. Previous work has shownn that olfactory ethanol attraction depends on the reinforcement of the olfactory information by a subset of octopaminergic/tyraminergic neurons and the aversion depends on the activation of a subset of dopaminergic neurons acting as a negative reinforcer. This study shows that the maintenance of olfactory information by the serotonergic CSD neurons promote the approach to the ethanol food odor mixture. In the presence of prolonged external olfactory information, the inhibitory information of an internal serotonergic reference system is overruled. The regulation of behavioral outcome requires a tight temporal regulation between the positive and negative reinforcing, inhibiting and prolonging mechanisms depending on the location of the fly relative to the ethanol containing food source and its internal condition. It will be interesting to see where the four mechanisms intersect with each other, how they are orchestrated and whether they are sufficient to regulate the attraction to ethanol. Furthermore, given the evolutionarily conserved nature of the serotonin transporter and the wide expression of serotonin, the serotonergic gating mechanism might delineate a basic principle for other behaviors and organisms as well (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).

    Unveiling the dual role of the dopaminergic system on locomotion and the innate value for an aversive olfactory stimulus in Drosophila

    The communication between sensory systems and the specific brain centers that process this information is crucial to develop adequate behavioral responses. Modulatory systems, including dopaminergic circuits, regulate this communication to finely tune the behavioral response associated to any given stimulus. For instance, the Mushroom Body (MB), an insect brain integration center that receives and processes several sensory stimuli and organizes the execution of motor programs, communicates with MB output neurons (MBONs) to develop behavioral responses associated to olfactory stimuli. This communication is modulated by dopaminergic neural systems. This study shows that silencing dopaminergic neurons increases the aversive response observed in adult flies exposed to Benzaldehyde (Bz) or octanol. The contribution was studied of two dopaminergic clusters that innervate different zones of MB, Protocerebral anterior medial (PAM) and Protocerebral posterior lateral 1 (PPL1), on the innate value to the aversive stimulus and the associated locomotor behavior. In order to do this, the synaptic transmission of these neural clusters was manipulated through the expression of Tetanus toxin, Kir2.1 and Transient receptor potential cation channel A1 (TrpA1) channels. The results show that neurons in PPL1 and PAM differentially modulate the innate value to Bz in adult flies. On the other hand, blocking neurotransmission or genetic silencing of PAM neurons results in decreased locomotor behavior in flies, an effect not observed when silencing PPL1. These results suggest that as in mammals, specific dopaminergic pathways differentially modulate locomotor behavior and the innate value for an odorant, a limbic-like response in Drosophila (Fuenzalida-Uribe, 2017).

    Recurrent architecture for adaptive regulation of learning in the insect brain

    Dopaminergic neurons (DANs) drive learning across the animal kingdom, but the upstream circuits that regulate their activity and thereby learning remain poorly understood. This study provides a synaptic-resolution connectome of the circuitry upstream of all DANs in a learning center, the mushroom body of Drosophila larva. Afferent sensory pathways and a large population of neurons were discovered that provide feedback from mushroom body output neurons and link distinct memory systems (aversive and appetitive). This was combined with functional studies of DANs and their presynaptic partners and with comprehensive circuit modeling. It was found that DANs compare convergent feedback from aversive and appetitive systems, which enables the computation of integrated predictions that may improve future learning. Computational modeling reveals that the discovered feedback motifs increase model flexibility and performance on learning tasks. This study provides the most detailed view to date of biological circuit motifs that support associative learning (Eschbach, 2020).

    To behave adaptively in an ever-changing environment, animals must be able to learn new associations between sensory cues (conditioned stimuli, CS) and rewards or punishments (aversive and appetitive unconditioned stimuli, US), and continuously update previous memories, depending on their relevance and reliability (Eschbach, 2020).

    Modulatory neurons (for example, DANs) convey information about rewards and punishments, and provide the teaching signals for updating the valence associated with CS in learning circuits across the animal kingdom (for example, the vertebrate basal ganglia or the insect mushroom body, MB). The co-occurrence of CS and modulatory neuron activity tuned only to the received US can support simple associative memory formation. To account for more complex behavioral phenomena, theories have been developed in which learning can be regulated by previously formed associations. According to reinforcement learning theories, learning is driven by errors between predicted and actual US (prediction errors) which are thought to be represented by the activity of DANs. Indeed, in many model organisms, the responses of modulatory neurons have been shown to be adaptive, including monkeys, rodents and insects. Despite recent progress the basic principles by which DAN activity is adaptively regulated and teaching signals are computed are not well understood (Eschbach, 2020).

    A prerequisite for the adaptive regulation of modulatory neuron activity is convergence of afferent pathways that convey information about received US with feedback pathways that convey information about previous experiences. A comprehensive synaptic-resolution connectivity map of the feedback circuits that regulate modulatory neurons would provide a basis for understanding how learning is adaptively regulated by prior learning, but it has previously been out of reach (Eschbach, 2020).

    Insects, especially in their larval stages, have small and compact brains that have recently become amenable to large-scale electron microscopy (EM) circuit mapping. Both adult and larval insect stages possess a brain center that is essential for associative learning, the MB. The MB contains neurons called Kenyon cells (KCs) that sparsely encode CS, MB modulatory neurons (collectively called MB input neurons, MBINs) that provide the teaching signals and MB output neurons (MBONs) whose activity represents learnt valences of stimuli. In the Drosophila larva, most modulatory neurons are DANs, some are octopaminergic neurons (OANs) and some have unidentified neurotransmitters (simply called MBINs). Modulatory neurons and MBONs project axon terminals and dendrites, respectively, onto the KC axons in a tiled manner, defining MB compartments, in both adult and larval Drosophila. In adult Drosophila, it has been shown that coactivation of KCs and DANs reduces the strength of the KC-MBON synapse in that compartment. Different compartments have been implicated in the formation of distinct types of memories, for example, aversive and appetitive, or short and long term. However, despite a good understanding of the structure and function of the core components of the MB in both adult and larval Drosophila, the circuits presynaptic to modulatory neurons that regulate their activity have remained relatively uncharacterized (Eschbach, 2020).

    This study therefore reconstructed all neurons presynaptic to all modulatory neurons in an EM volume that spans the entire nervous system of a first instar Drosophila larva, in which all the core components of the MB were previously reconstructed. This study also determined which individual modulatory neurons are activated by punishments and reconstructed their afferent US pathways from nociceptive and mechanosensory neurons. The neurotransmitter profiles of some of the neurons in the network were characterized and some of the identified structural connections were functionally confirmed. Finally, a model was developed of the circuit constrained by the connectome, the neurotransmitter data and the functional data, and it was used to explore the computational advantages offered by the recently discovered architectural motifs for performing distinct learning tasks (Eschbach, 2020).

    Modulatory neurons (for example, DANs) are key components of higher-order circuits for adaptive behavioral control, and they provide teaching signals that drive memory formation and updating. This study provides a synaptic-resolution connectivity map of a recurrent neural network that regulates the activity of modulatory neurons in a higher-order learning center, the Drosophila larval MB. Some of the recently identified structural pathways were functionally tested, and a model of the circuit was developed to explore the roles of these motifs in different learning tasks (Eschbach, 2020).

    A large population was discovered of 61 feedback neuron pairs that provide one- and/or two-step feedback from the MBONs to modulatory neurons. Strikingly, it was found that many modulatory neurons receive more than 50% of their total dendritic input from feedback pathways. These results suggest that prior memories as represented by the pattern of MBON activity can strongly influence modulatory neuron activity (Eschbach, 2020).

    Learning and memory systems in vertebrates and insects are often organized into distinct compartments implicated in forming distinct types of memories (for example, aversive and appetitive or short and long term). Interestingly, it was found that the majority of the discovered feedback pathways link distinct memory systems, suggesting that the MB functions as an interconnected ensemble during learning. Thus, prior memories formed about an odor in one compartment can influence the formation and updating of distinct types of memories about that odor in other compartments (Eschbach, 2020).

    In adult Drosophila, functional connections between some MBONs and DANs have been reported, and some have been shown to play a role in short-term memory formation, long-term memory consolidation, extinction and reconsolidation, or in synchronizing DAN ensemble activity in a context-dependent manner. In some cases, direct MBON-to-DAN connections have been demonstrated. Although direct connections from several MBONs onto DANs exist in the larva, this study found that indirect connections via the feedback neurons account for a much larger fraction of a modulatory neuron's dendritic input than direct MBON synapses. This suggests that adaptive DAN responses may be largely driven by such indirect feedback (Eschbach, 2020).

    Some of the one-step within-compartment feedback motifs that were found are analogous to the feedback motifs so far described for the DANs in the vertebrate midbrain. Although the diversity and the inputs of striatal feedback neurons have not yet been fully explored, in the future it will be interesting to determine whether many of the striatal feedback neurons also link distinct memory systems (Eschbach, 2020).

    The use of internal predictions can dramatically increase the flexibility of a learning system. This study reveals candidate circuit motifs that could compute integrated predicted value signals across appetitive and aversive memory systems. A prominent motif that was identified is convergence of excitatory and inhibitory connections from MBONs from compartments of opposite valence onto DANs. In naive animals, odor-evoked MBON excitation in all compartments is thought to be similar. However, associative learning selectively depresses conditioned odor drive to MBONs in compartments where modulatory neuron activation has been paired with the odor. It is proposed that by comparing the conditioned odor-evoked MBON excitation in compartments of opposite valence via cross-compartment feedback connections, modulatory neurons compute an integrated predicted value signal across appetitive and aversive domains (Eschbach, 2020).

    Convergence of feedback and US pathways could allow the computation of prediction errors An important aspect of reinforcement learning theories is the idea that modulatory neurons compare predicted and actual US (to compute so-called prediction errors) and drive memory formation or extinction depending on the sign of the prediction error. Although Drosophila modulatory neurons have not yet been directly shown to represent prediction errors, adult and larval Drosophila are capable of extinction, and the current study reveals candidate motifs that could support the comparison of expected and actual US. Modulatory neurons were found to receive convergent input from feedback pathways from MBONs and from US pathways. Modulatory neurons could therefore potentially compute prediction errors by comparing inhibitory drive from the feedback pathways with the excitatory drive from the US pathways, or vice versa. Consistent with this idea, some DANs were observed in this model that are inhibited by US alone and activated by CS+ alone, or vice versa (Eschbach, 2020).

    This study also reveals that US pathways and feedback pathways converge at two levels: not only at the modulatory neurons themselves, but also at the FB2Ns. Actual and expected outcomes could therefore also be compared by FB2Ns. A recent study in the mouse ventral tegmental area has found that some pre-DAN neurons encoded only actual or only expected reward, whereas others encoded both variables. Thus, both in vertebrates and in insects, comparing predicted and actual outcomes may be a complex computation involving multiple levels of integration that eventually converge onto an ensemble of modulatory neurons (Eschbach, 2020).

    An assumption in many reinforcement learning models is that all modulatory neurons receive a global scalar reward prediction error signal. The current study was able to analyze the comprehensive set of inputs of every individual uniquely identifiable modulatory neuron in a learning center. This revealed that each modulatory neuron receives a unique set of feedback inputs that could enable each neuron to compute a unique set of features. Consistent with this, a diversity of adaptive response types in the modulatory neurons was observed in this model This suggests that instead of computing a single global reward prediction error that is distributed to all modulatory neurons, the network uses a range of distinct compartmentalized and distributed teaching signals (Eschbach, 2020).

    The connectivity and modeling studies revealed two architectural features of the circuit that provide input to the modulatory neurons that increase its performance and flexibility on learning tasks. The first is the multilevel feedback architecture that includes not only the previously known direct MBON feedback, but also multiple levels of indirect feedback. The second is the extensive set of cross-compartment connecti,.ons. Modeling suggests that these motifs support improved performance on complex tasks that require the computation of variables such as predictions, prediction errors and context (Eschbach, 2020).

    In summary, this study presents a complete circuit diagram of a recurrent network that computes teaching signals in a biological system, providing insights into the architectural motifs that increase its computational power and flexibility. The connectome-constrained model provides numerous predictions that can be tested in the future in a tractable model organism, for which genetic tools can be generated to monitor and manipulate individual neurons. The connectome, together with the functional and modeling studies, therefore provides exciting opportunities for elucidating the biological implementation of reinforcement learning algorithms (Eschbach, 2020).

    Tyramine action on motoneuron excitability and adaptable tyramine/octopamine ratios adjust Drosophila locomotion to nutritional state

    Adrenergic signaling profoundly modulates animal behavior. For example, the invertebrate counterpart of norepinephrine, octopamine, and its biological precursor and functional antagonist, tyramine, adjust motor behavior to different nutritional states. In Drosophila larvae, food deprivation increases locomotor speed via octopamine-mediated structural plasticity of neuromuscular synapses, whereas tyramine reduces locomotor speed, but the underlying cellular and molecular mechanisms remain unknown. This study shows that tyramine is released into the CNS to reduce motoneuron intrinsic excitability and responses to excitatory cholinergic input, both by tyramine(honoka) receptor activation and by downstream decrease of L-type calcium current. This central effect of tyramine on motoneurons is required for the adaptive reduction of locomotor activity after feeding. Similarly, peripheral octopamine action on motoneurons has been reported to be required for increasing locomotion upon starvation. It was further shown that the level of tyramine-beta-hydroxylase (TBH), the enzyme that converts tyramine into octopamine in aminergic neurons, is increased by food deprivation, thus selecting between antagonistic amine actions on motoneurons. Therefore, octopamine and tyramine provide global but distinctly different mechanisms to regulate motoneuron excitability and behavioral plasticity, and their antagonistic actions are balanced within a dynamic range by nutritional effects on TBH (Schutzler, 2019).

    AANAT1 functions in astrocytes to regulate sleep homeostasis

    How the brain controls the need and acquisition of recovery sleep after prolonged wakefulness is an important issue in sleep research. The monoamines serotonin and dopamine are key regulators of sleep in mammals and in Drosophila. This study found that the enzyme arylalkylamine N-acetyltransferase 1 (AANAT1) is expressed by Drosophila astrocytes and specific subsets of neurons in the adult brain. AANAT1 acetylates monoamines and inactivates them, and it was found that AANAT1 limited the accumulation of serotonin and dopamine in the brain upon sleep deprivation. Loss of AANAT1 from astrocytes, but not from neurons, caused flies to increase their daytime recovery sleep following overnight sleep deprivation. Together, these findings demonstrate a crucial role for AANAT1 and astrocytes in the regulation of monoamine bioavailability and homeostatic sleep (Davla, 2020).

    Characteristic features of sleep are conserved among species, and from humans to insects sleep is influenced by neural circuits involving monoamines such as serotonin and dopamine. Glial cells are known to take up and metabolize monoamines and they have been increasingly implicated in mechanisms of baseline and homeostatic sleep regulation in mammals and flies, but it remains unknown whether and how glia might influence monoaminergic control of sleep. Sleep is regulated by circadian clocks and a homeostatic drive to compensate for prolonged wakefulness, and growing evidence suggests that neural mechanisms controlling homeostatic sleep can be discriminated from those controlling baseline sleep. In Drosophila, mutants of arylalkylamine N-acetyltransferase 1 (AANAT1lo) were reported to have normal baseline amounts of sleep and motor activity, but increased recovery sleep ('rebound') following deprivation. AANAT1 corresponds to speck, a long-known mutation characterized by a darkly pigmented region at the wing hinge. AANAT1 can acetylate and inactivate monoamines in vitro, but the role of AANAT1 in vivo remains poorly understood (Davla, 2020).

    This paper reports that AANAT1 is expressed in astrocytes and subsets of neurons in the adult Drosophila brain, with levels in astrocytes first rising then declining markedly overnight. In sleep deprived AANAT1 mutant flies, heightened recovery sleep is accompanied by increased serotonin and dopamine levels in the brain. With cell type selective AANAT1 knockdown, this study found that AANAT1 functions in astrocytes, but not neurons, to limit the amount of recovery sleep that flies take in response to sleep deprivation (SD). These findings identify a critical role for astrocytes in the regulation of monoamine bioavailability and calibration of the response to sleep need (Davla, 2020).

    These findings illustrate a newly discovered role for astrocytes in the control of monoamine bioavailability and homeostatic sleep drive, where they are specifically engaged to catabolize monoamines whose levels are elevated by overnight SD. Drosophila astrocytes also express the enzyme Ebony, which couples dopamine to N-β-alanine, and a receptor for octopamine and tyramine, reinforcing how they are well-equipped to metabolize monoamines, and to monitor and respond to monoaminergic neuronal activity. Neither gene expression studies nor RNA sequencing databases provide evidence for monoamine-synthesizing enzymes in Drosophila astrocytes, so it appears likely that monoamines inactivated by AANAT1 in astrocytes are brought into these cells by an unidentified transporter. Astrocytes are particularly well-suited for regulating sleep in this way because they have ramified processes that infiltrate neuropil regions to lie in close proximity to synapses. SD can seemingly reduce the degree of contact between astrocytes and neurons in the fly brain, and so it is possible that these structural changes could influence monoamine uptake and inactivation by astrocytes (Davla, 2020).

    In neurons, AANAT1 may function to limit sleep consolidation at night, but evidence for this came from only one of the two RNAi lines used in this study and was not observed in AANAT1lo mutants. Further studies are needed to characterize sleep-control functions of AANAT1 in neurons, if any, to understand better how cellular context can impact AANAT1 function in sleep regulation. In light of this, it is noted that loss of the related enzyme aanat2 in zebrafish larvae decreases baseline sleep, which could be attributed to a loss of melatonin since the AANAT1 product N-acetylserotonin is an intermediate in the synthesis of melatonin in vertebrates. Clearly, the appropriate balance and cellular context of AANAT activity is critical for the regulation of sleep, and this study shows in Drosophila that astrocytes are an important contributor to this balance. Interestingly, astrocytes in rodents express the monoamine transporters and receptors for dopamine and serotonin, raising the possibility that astrocytes in mammals might also participate in mechanisms of sleep regulation involving monoaminergic neural signaling (Davla, 2020).

    Neuronal octopamine signaling regulates mating-induced germline stem cell increase in female Drosophila melanogaster

    Stem cells fuel the development and maintenance of tissues. Many studies have addressed how local signals from neighboring niche cells regulate stem cell identity and their proliferative potential. However, the regulation of stem cells by tissue-extrinsic signals in response to environmental cues remains poorly understood. This study reports that efferent octopaminergic neurons projecting to the ovary are essential for germline stem cell (GSC) increase in response to mating in female Drosophila. The neuronal activity of the octopaminergic neurons is required for mating-induced GSC increase as they relay the mating signal from sex peptide receptor-positive cholinergic neurons. Octopamine and its receptor Oamb are also required for mating-induced GSC increase via intracellular Ca(2+) signaling. Moreover, Matrix metalloproteinase-2 was identified as a downstream component of the octopamine-Ca(2+) signaling to induce GSC increase. This study provides a mechanism describing how neuronal system couples stem cell behavior to environmental cues through stem cell niche signaling (Yoshinari, 2020).

    This study reports that the mating-induced GSC increase in female D. melanogaster is regulated by octopaminergic neurons directly projecting to the ovary. From in vivo and ex vivo experiments, the following model is proposed to explain the mating-induced GSC increase. After mating, the male seminal fluid SP is transferred into the female uterus, stimulating SPR-positive neurons. As the liganded SPR silences the neuronal activity of SPR-positive sensory neurons (SPSNs), the acetylcholine released from SPSNs is suppressed. As SPSNs and dsx+ Tdc2+ neurons are directly connected, this suppression directly modulates dsx+ Tdc2+ neuronal activity. Because this study has shown that nAChRs in dsx+ Tdc2+ neurons exhibit an inhibitory effect with an unknown mechanism, the inactivation of nAChRs in the absence of acetylcholine results in the activation of dsx+ Tdc2+ neurons in mated females. As a consequence, octopamine is released from dsx+ Tdc2+ neurons, received by Oamb, induces [Ca2+]i in the escort cells, and finally activates the Mmp2 enzymatic activity. The activity of Mmp2 positively regulates the Dpp-mediated niche signaling, thereby leading to mating-induced GSC increase. (Yoshinari, 2020).

    The proposed model is that the OA from dsx+ Tdc2+ neurons is directly received by the escort and follicle cells in the germarium. This model is supported by two observations. First, mating-induced GSC increase is impaired by Oamb RNAi using a GAL4 driver that is active specifically in the germarium cells but not mature follicle cells. Second, OA treatment evokes [Ca2+]i elevation in these germarium cells in an Oamb-dependent manner. However, this study did not address whether the escort cells and/or the follicle cells in the germarium express Oamb, as no clear GAL4 expression was observed in two independent Oamb-T2A-GAL4 drivers. It is surmised that this may be due to lower amounts of Oamb transcript in the germarium (Yoshinari, 2020).

    It was shown that the activation of the ovary-projecting dsx+ Tdc2+ neurons is necessary and sufficient to induce GSC increase. However, from an anatomical point of view, the dsx+ Tdc2+ neurons project to the distal half of the ovary but not to the germarium. Considering the model described above, this disagreement can be attributed to the characteristic volume transmission of monoamine neurotransmitters. In other words, neurotransmitters act at a distance well beyond their release sites from cells or synapses. Therefore, the OA secreted from the terminals of dsx+ Tdc2+ neurons could reach the germarium located at the most proximal part of the ovary (Yoshinari, 2020).

    Several previous studies have revealed that OA signaling has a pivotal role in reproductive tissues other than germarium, such as mature follicle cells, oviduct, and ovarian muscle, to promote ovulation, oviduct remodeling, and ovarian-muscle contraction, respectively. Therefore, it is likely that the dsx+ Tdc2+ neurons orchestrate multiple different events during oogenesis in response to mating stimulus. Because a mated female needs to activate oogenesis to continuously produce eggs in concert with sperm availability, it is reasonable that the ovary-projecting neurons switch on the activity of the entire process of reproduction (Yoshinari, 2020).

    Based on the present study and several previous studies, the OA-Oamb-Ca2+-Mmp2 axis is required for GSC increase and follicle rupture, both of which are induced by mating stimuli in D. melanogaster. In both cases, Mmp2 enzymatic activity is likely to be essential, as the overexpression of Timp encoding a protein inhibitor of Mmp2 suppresses GSC increase, as well as follicle rupture. Mmp2 in mature follicle cells cleaves and downregulates Viking/collagen VI. In fact, several previous studies have revealed that Viking/collagen VI is required for GSC maintenance in female D. melanogaster. However, no significant change was observed in Viking/Collagen VI levels in the germarium between the control and Mmp2 RNAi flies. Therefore, it is concluded that Viking/collagen VI is not a substrate of Mmp2 in the regulation of mating-induced GSC increase. Besides Viking/Collagen VI, Dally-like (Dlp) is another basement membrane protein associated with extracellular matrix and known as the Mmp2 substrate. Interestingly, dlp is expressed in the escort cells. Moreover, Dlp controls the distribution of Dpp and Wnts, both of which significantly affect GSC self-renewal and differentiation. Future research should decipher the exact substrate by which Mmp2 controls Dpp and/or Wnts to modulate GSC behavior in response to mating stimulus (Yoshinari, 2020).

    Another remaining question to be addressed is how Mmp2 function is regulated in GSC increase. Ecdysteroid biosynthesis and signaling in the ovary are necessary but not sufficient for the OA-Oamb-Ca2+-mediated GSC increase and follicle rupture. This study found that in the regulation of mating-induced GSC increase, ecdysteroid signaling acts downstream of Ca2+ signaling. On the other hand, in the follicle rupture process, ecdysteroid signaling either acts downstream, upstream, or both, of Ca2+ signaling. Further, the precise action of ecdysteroid has yet to be elucidated. The Mmp2-GFP fusion protein level in the follicle cells is not changed in the loss-of-Ecdysone receptor-function flies, implying that ecdysteroid signaling might regulate Mmp2 enzymatic activity by an unknown mechanism. Considering the involvement of both the OA-Oamb-Ca2+-Mmp2 axis and ecdysteroid biosynthesis, it is very likely that the Mmp2 enzymatic activity is also regulated by the same, unknown mechanism not only in the mature follicle cells to control follicle rupture, but also in the germarium to control mating-induced GSC increase (Yoshinari, 2020).

    In many animals, reproduction involves significant behavioral and physiological shifts in response to mating. In female D. melanogaster, several post-mating responses are coordinated by SPSNs and their downstream afferent neuronal circuit, including Stato-Acoustic Ganglion neurons, the ventral abdominal lateral Myoinhibitory peptide neurons, and the efferent dsx+ Tdc2+ neurons. GRASP analysis indicates a direct synaptic connection between cholinergic SPSNs and OAergic neurons. Moreover, this study demonstrated that nAChRs in dsx+ Tdc2+ neurons are responsible for the suppression of their neuronal activity in virgin females. However, nAChRs are the cation channels leading to depolarization upon acetylcholine binding, and therefore usually activate neurons. How is the opposite role of nAChRs in dsx+ Tdc2+ neuronal activity achieved? One possibility is that acetylcholine-nAChR signaling does not evoke a simple depolarization but rather generates a virgin-specific temporal spike pattern in dsx+ Tdc2+ neurons. Interestingly, recent studies demonstrated that the pattern, instead of the frequency, of neuronal firing is significant in adjusting the neuronal activity of clock neurons in D. melanogaster. The firing pattern relies on control of ionic flux by the modulation of Ca2+-activated potassium channel and Na+/K+ ATPase activity. Because whether mating changes the firing pattern of dsx+ Tdc2+ neurons remains to be examined, the neuronal activity in SPSNs and the dsx+ Tdc2+ neuronal circuit between virgin and mated females are future research areas (Yoshinari, 2020).

    In the last decades, there is growing evidence that GSCs and their niche are influenced by multiple humoral factors. Based on the data from the current study and previous studies, there are at least four crucial humoral factors for regulating the increase and/or maintenance of D. melanogaster female GSCs, including DILPs, ecdysteroids, Neuropeptide F (NPF), and OA. Notably, all of these come from different sources: DILPs are from the insulin-producing cells located in the pars intercerebralis of the central brain; ecdysteroids from the ovary; NPF from the midgut; and OA from the neurons located in the abdominal ganglion. In addition to these identified humoral factors, recent studies also imply that adiponectin and unknown adipocyte-derived factor(s) are essential for GSC maintenance. These data clearly indicate that D. melanogaster female GSCs are systemically regulated by interorgan communication involving multiple organs. The additional interorgan communication mechanisms that ensure the faithful coupling of the increase and maintenance of GSC to the organism's external and physiological environments are essential to be investigated in future studies (Yoshinari, 2020).

    To modulate the increase and maintenance of GSC, ecdysteroids are received by both GSCs and niche cells, whereas DILPs, NPF, and OA are received by niche cells. A major signal transduction mechanism of each of these humoral factors have been well characterized, namely phosphoinositide 3-kinase pathway for DILPs-InR signaling, EcR/Ultraspiracle-mediated pathway for ecdysteroid signaling, cAMP pathway for NPF-NPFR signaling, and Ca2+ pathway for OA-Oamb signaling. However, it remains unclear whether and how each of these signaling pathways control the production and secretion of the niche signal, as well as its distribution and transduction. In addition, it is important to understand whether and how the multiple system signals are integrated to control the mating-induced increase and maintenance of GSCs (Yoshinari, 2020).

    In recent years, many studies have revealed that not only local niche signals but also systemic and neuronal factors play indispensable roles in regulating GSC behavior. In D. melanogaster, ecdysteroid signaling is essential for the proliferation and maintenance of GSCs and neural stem cells. This study has identified the ovary-projecting OAergic neurons as new regulators of stem cell homeostasis. Both steroid hormones and OA-like monoamines, such as noradrenaline, are also involved in stem cell regulation in mammals. For example, the mammalian steroid hormone, estrogen, is important in regulating cell division and/or maintenance of hematopoietic stem cells, mammary stem cell, neural stem cells, and hematopoietic stem cells. Moreover, noradrenergic neurons, which directly project to the bone marrow, regulate the remodeling of hematopoietic stem cells niche. Therefore, the steroid hormone- and noradrenergic nerve-dependent control of stem cell homeostasis are likely conserved across animal species. In this regard, the D. melanogaster reproductive system will further serve as a powerful model to unravel the conserved systemic and neuronal regulatory mechanisms for stem cell homeostasis in animals (Yoshinari, 2020).

    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)

    Serotonergic modulation of aggression in Drosophila involves GABAergic and cholinergic opposing pathways

    Pathological aggression is commonly associated with psychiatric and neurological disorders and can impose a substantial burden and cost on human society. Serotonin (5HT) has long been implicated in the regulation of aggression in a wide variety of animal species. In Drosophila, a small group of serotonergic neurons selectively modulates the escalation of aggression. This study has identified downstream targets of serotonergic input-two types of neurons with opposing roles in aggression control. The dendritic fields of both neurons converge on a single optic glomerulus LC12, suggesting a key pathway linking visual input to the aggression circuitry. The first type is an inhibitory GABAergic neuron: its activation leads to a decrease in aggression. The second neuron type is excitatory: its silencing reduces and its activation increases aggression. RNA sequencing (RNA-seq) profiling of this neuron type identified that it uses acetylcholine as a neurotransmitter and likely expresses 5HT1A, short neuropeptide F receptor (sNPFR), and the resistant to dieldrin (RDL) category of GABA receptors. Knockdown of RDL receptors in these neurons increases aggression, suggesting the possibility of a direct crosstalk between the inhibitory GABAergic and the excitatory cholinergic neurons. These data show further that neurons utilizing serotonin, GABA, ACh, and short neuropeptide F interact in the LC12 optic glomerulus. Parallel cholinergic and GABAergic pathways descending from this sensory integration area may be key elements in fine-tuning the regulation of aggression (Alekseyenko, 2019).

    Octopamine neuron dependent aggression requires dVGLUT from dual-transmitting neurons

    Neuromodulators such as monoamines are often expressed in neurons that also release at least one fast-acting neurotransmitter. The release of a combination of transmitters provides both 'classical' and 'modulatory' signals that could produce diverse and/or complementary effects in associated circuits. This study establishes that the majority of Drosophila octopamine (OA) neurons are also glutamatergic and identifed the individual contributions of each neurotransmitter on sex-specific behaviors. Males without OA display low levels of aggression and high levels of inter-male courtship. Males deficient for dVGLUT solely in OA-glutamate neurons (OGNs) also exhibit a reduction in aggression, but without a concurrent increase in inter-male courtship. Within OGNs, a portion of VMAT and dVGLUT puncta differ in localization suggesting spatial differences in OA signaling. These findings establish a previously undetermined role for dVGLUT in OA neurons and suggests that glutamate uncouples aggression from OA-dependent courtship-related behavior. These results indicate that dual neurotransmission can increase the efficacy of individual neurotransmitters while maintaining unique functions within a multi-functional social behavior neuronal network (Sherer, 2020).

    Addressing the functional complexities of 'one neuron, multiple transmitters' is critical to understanding how neuron communication, circuit computation, and behavior can be regulated by a single neuron. Over many decades, significant progress has been made elucidating the functional properties of neurons co-expressing neuropeptides and small molecule neurotransmitters, where the neuropeptide acts as a co-transmitter and modulates the action of the neurotransmitter. Only recently have studies begun to examine the functional significance of co-transmission by a fast-acting neurotransmitter and a slow-acting monoamine (Sherer, 2020).

    This study has demonstrated that OA neurons express dVGLUT and has utilized a new genetic tool to remove dVGLUT in OA-glutamate neurons. Quantifying changes in the complex social behaviors of aggression and courtship revealed that dVGLUT in brain OGNs is required to promote aggressive behavior and a specific behavioral pattern, the lunge. In contrast, males deficient for dVGLUT function do not exhibit an increase in inter-male courtship. These results establish a previously undetermined role for dVGLUT in OA neurons located in the adult brain and reveal glutamate uncouples aggression from inter-male courtship. It has been suggested that classical neurotransmitters and monoamines present in the same neuron modulate each other's packaging into synaptic vesicles or after release via autoreceptors. For example, a reduction of dVGLUT in DA-glutamate neurons resulted in decreased AMPH-stimulated hyperlocomotion in Drosophila and mice suggesting a key function of dVGLUT is the mediation of vesicular DA content. In this study, the independent behavioral changes suggests enhancing the packaging of OA into vesicles is not the sole function of dVGLUT co-expression and suggests differences in signaling by OA from OGNs on courtship-related circuitry (Sherer, 2020).

    Co-transmission can generate distinct circuit-level effects via multiple mechanisms. One mechanism includes spatial segregation; the release of two neurotransmitters or a neurotransmitter and monoamine from a single neuron occurring at different axon terminals or presynaptic zones. Recent studies examining this possible mechanism have described (1) the release of GLU and DA from different synaptic vesicles in midbrain dopamine neurons and (2) the presence of VMAT and VGLUT microdomains in a subset of rodent mesoaccumbens DA neurons. This study expressed a new conditionally expressed epitope-tagged version of VMAT in OGNs and visualized endogenous dVGLUT via antibody labeling. Within OGNs, the colocalization of VMAT and dVGLUT puncta was not complete suggesting the observed behavioral phenotype differences may be due to spatial differences in OA signaling (Sherer, 2020).

    A second mechanism by which co-transmission may generate unique functional properties relies on activating distinct postsynaptic receptors. In Drosophila, recent work has identified a small population of male-specific neurons that express the alpha-like adrenergic receptor, OAMB, as aggression-promoting circuit-level neuronal targets of OA modulation independent of any effect on arousal and separately knockdown of the Rdl GABAa receptor in a specific doublesex+ population stimulated male aggression (Watanabe, 2017). Future experiments identifying downstream targets that express both glutamate and octopamine receptors would be informative, as well as using additional split-Gal4 lines to determine if segregation of transporters is a hallmark of the majority of OGNs. Finally, a third possible mechanism is Glu may be co-released from OGNs and act on autoreceptors to regulate presynaptic OA release (Sherer, 2020).

    Deciphering the signaling complexity that allows neural networks to integrate external stimuli with internal states to generate context-appropriate social behavior is a challenging endeavor. Neuromodulators including monoamines are released to signal changes in an animal's environment and positively or negatively reinforce network output. In invertebrates, a role for OA in responding to external chemosensory cues as well as promoting aggression has been well-established. In terms of identifying specific aggression circuit-components that utilize OA, previous results determined OA neurons directly receive male-specific pheromone information and the aSP2 neurons serve as a hub through which OA can bias output from a multi-functional social behavior network towards aggression. The ability of OA to bias behavioral decisions based on positive and negative reinforcement was also recently described for food odors. In vertebrates, it has been proposed that DA-GLU cotransmission in the NAc medial shell might facilitate behavioral switching. The finding that the majority of OA neurons are glutamatergic, suggests that the complex social behavior of aggression may rely on small subsets of neurons that both signal the rapid temporal coding of critical external stimuli as well as the frequency coding of such stimuli resulting in the enhancement of this behavioral network. One implication of the finding regarding the separable OA-dependent inhibition of inter-male courtship is the possibility of identifying specific synapses or axon terminals that when activated gate two different behavioral outcomes. A second implication is that aggressive behavior in other systems may be modified by targeting GLU function in monoamine neurons (Sherer, 2020).

    Finally, monoamine-expressing neurons play key roles in human behavior including aggression and illnesses that have an aggressive component such as depression, addiction, anxiety, and Alzheimer's. While progress is being made in addressing the functional complexities of dual transmission, the possible pathological implications of glutamate co-release by monoamine neurons remains virtually unknown. Analyzing the synaptic vesicle and release properties of monoamine-glutamate neurons could offer new possibilities for therapeutic interventions aimed at controlling out-of-context aggression (Sherer, 2020).

    A circuit node that integrates convergent input from neuromodulatory and social behavior-promoting neurons to control aggression in Drosophila

    Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. A systematic, unbiased screen identified OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. The results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM(+) neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals (Watanabe, 2017).

    A rich behavioral literature has implicated OA in the control of invertebrate aggression, although the direction of its effects differs between species. Classic studies in lobsters have shown that injection of OA into the hemolymph promotes a subordinate-like posture, while injection of serotonin (5HT) produces a dominant-like posture. In contrast, hemolymph injections of OA in crickets restore aggressiveness to subordinated animals, mimicking the arousing effects of episodes of free flight. OA has also been suggested to play a role in aggressive motivation restored to defeated crickets by residency in a shelter. In Drosophila, null mutations of TβH strongly suppressed aggressiveness, suggesting a positive-acting role for OA in flies as in crickets. Interestingly, intra-hypothalamic infusion of NE in mammals can also enhance aggression. However, little is known about the neurons on which these amines act directly to influence aggression, in any organism (Watanabe, 2017).

    This study applied a novel, unbiased approach to identify OARNs relevant to aggression in Drosophila. Importantly this screen was based not on mutations in OAR genes, but rather upon genetic silencing of neurons that express GAL4 under the control of different OAR gene cis-regulatory modules (CRMs). This screen was agnostic with respect to which OAR gene is involved, or in which neurons that OAR is expressed. It yielded a small population of male-specific, FruM+ OA-sensitive neurons, called aSP2, the activity of which is required for normal levels of aggressiveness. No significant change in UWEs (male-male courtship) was observed when these neurons were activated or silenced. Nevertheless, neuronal silencing in the parental R47A04-GAL4 line increased male-male courtship, perhaps reflecting an inhibitory role for non-aSP2 neurons in that line. Therefore, while it is not possible to completely exclude a role for aSP2 neurons to suppress male-male courtship, the evidence does not strongly support it (Watanabe, 2017).

    Multiple lines of evidence suggest that R47A04aSP2 neurons are indeed OA responsive, likely via OAMB. First, these neurons are labeled by a CRM from the Oamb gene. Second, RNAi-mediated knockdown of Oamb in R47A04 neurons reduced aggression, phenocopying the effects of an Oamb null allele. (However, knockdown using the split-GAL4 R47A04aSP2driver only yielded a trend to reduced aggression that did not reach significance, perhaps reflecting a floor effect in this assay.) Third, overexpression of Oamb cDNAs in these neurons using R47A04-GAL4 rescued the Oamb null mutant and enhanced the effect of OA feeding to promote aggression. Fourth,R47A04aSP2 neurons were activated by bath-applied OA in brain explants, and this effect was also blocked by RNAi-mediated knockdown of Oamb. Taken together, these data strongly suggest that aSP2 neurons respond directly to OA to mediate its effects on aggression, although they do not exclude a role for other OA-responsive non-aSP2 neurons in line R47A04. While it was not possible to definitively establish which of the 27 different classes of OANs in Drosophila provide functional OA input to aSP2 cells, some candidate OA neurons labeled in retrograde PA-GFP experiments (VUM and VPM) have previously been implicated in aggression (Watanabe, 2017).

    In Drosophila OA, like NE in vertebrates, is thought to promote arousal. Consistent with such a function, OAergic fibers are broadly distributed across the entire Drosophila CNS, as are NE fibers in vertebrates. Thus OARNs could enhance aggression by increasing arousal, and there is evidence for such a function in crickets. However, manipulations of R47A04aSP2neurons that increased or decreased aggression did not affect locomotion, circadian activity, or sleep. This suggests that these neurons influence aggression directly and specifically, rather than by increasing generalized arousal. Other classes of OARNs not investigated in this study have been implicated in sleep-wake arousal (Watanabe, 2017).

    Does OA promote aggression in a permissive or instructive manner? While it is clear that OA synthesis and release are required for aggression in Drosophila, whether increasing OA suffices to promote aggression is less clear. It was reported that NaCh Bac-mediated activation of Tdc2-GAL4 neurons enhanced aggression, but the current study neither this manipulation, nor activation of Tdc2 neurons using dTrpA1 or Chrimson, yielded consistent effects. Thus, while OA is essential for normal levels of aggression, it is not clear whether it plays an instructive role to promote this behavior (Watanabe, 2017).

    In principle, OA RNs could act directly in command-like neurons that mediate aggression, or rather in cells that play a modulatory role. It was found that aggression was increased by tonically enhancing the excitability of R47A04aSP2 neurons using NaChBac, but not by phasically activating them optogenetically, arguing against a command-like function. Furthermore, the influence of TK FruM neurons, which do promote aggression when phasically activated, was not dependent on the activity of R47A04aSP2 neurons, indicating that the latter are not functionally downstream of the former. Together, these data argue against a role for R47A04aSP2 cells as command-like neurons, or as direct outputs of command neurons, for aggression. Rather, these cells exert a modulatory influence on agonistic behavior (Watanabe, 2017).

    In searching for neurons that may interact with R47A04aSP2 cells in their modulatory capacity, P1 neurons, a FruM+ population of 20 neurons/hemibrain was identified that controls male courtship, but which can also promote aggression when activated. It has been argued that this aggression-promoting effect is due to a subset of FruM neurons in the GAL4 line used in these studies, R71G01-GAL4. However, this study shows that conditional expression of FLP-ON Chrimson in a subset of neurons within the R71G01-GAL4 population using Fru-FLP. Nevertheless, these data do not exclude that the aggression-promoting neurons in the P1 cluster expressed Fru-FLP only transiently during development, nor do they exclude the possibility that different subpopulations of neurons within line R71G01 control courtship versus aggression; further studies will be required to resolve these issues (Watanabe, 2017).

    The P1 cluster is known to project to downstream cells that are specific for courtship . The present study provides the first evidence that cells in this cluster also functionally activate (and physically contact) aggression-specific neurons. However, due to limitations of the genetic reagents employed, it is not certain that the behavioral, physiological, and anatomical interactions with aSP2 cells demonstrated in this study are all mediated by the same subset of neurons in the P1 cluster. With this caveat in mind, these data suggest that aSP2 neurons are functionally downstream of both a subset(s) of P1 neurons, as well as of OA neurons (Watanabe, 2017).

    Feeding flies OA potentiated the activation of R47A04aSP2 neurons by P1 neuron stimulation, in brain explants. Furthermore, activation of aggression by P1 stimulation was enhanced and suppressed by pharmacologically increasing or decreasing OA signaling, respectively. While some off-target effects of the drugs, or an action on non-aSP2 neurons expressing OARs, cannot be excluded these pharmacologic effects were overridden by opposite-direction genetic manipulations of R47A04aSP2neuronal activity. Whether P1 neurons and OANs normally activate aSP2 neurons in vivo, simultaneously or sequentially, is not yet clear. Nevertheless it is striking that P1 and Tdc2 putative inputs occupy adjacent regions of aSP2 dendrites. Taken together, these findings suggest that aSP2 cells may serve as a node through which OA can bias output from a multifunctional social behavior network involving P1 neurons, in a manner that favors aggression. However, aSP2 neurons themselves do not appearto control directly the choice between mating and fighting (Watanabe, 2017).

    Male-specific cuticular hydrocarbons such as 7-tricosene (7-T) are known to be required for aggression. Interestingly, it has recently been shown that gustatory neurons expressing Gr32a, which encodes a putative 7-T receptor, innervate OANs in the SEZ; these OANs are activated by 7-T in a Gr32a-dependent manner. SEZ-innervating OANs include the VPM/VUM subsets seen in PA-GFP retrograde labeling experiments. These data raise the possibility that R47A04aSP2 neurons might be targets of VPM/VUM OANs activated by 7-T. If so, then they could provide a potential link between the influence of male-specific pheromones, OA, and central aggression circuitry. Studies of NE neurons in vertebrates have led to a prevailing view that this neuromodulator is released in a diffuse, 'sprinkler system'-like manner to control brain-wide states like arousal. Recent studies in Drosophila indicate that the broad, brain-wide distribution of OAergic fibers reflects the superposition of close to 30 anatomically distinct subclasses of OANs). The data presented in this study reveal a high level of circuit specificity for OARNs that mediate the effects of OA on aggression, mirroring the anatomical and functional specificity of OANs reported to control this behavior. If this anatomical logic is conserved, then such circuit specificity may underlie the actions of NE in mammals to a greater extent than is generally assumed (Watanabe, 2017).


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    Zygotically transcribed genes

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