Rewarding Capacity of Optogenetically Activating a Giant GABAergic Central-Brain Interneuron in Larval Drosophila.

Larvae of the fruit fly Drosophila melanogaster are a powerful study case for understanding the neural circuits underlying behavior. Indeed, the numerical simplicity of the larval brain has permitted the reconstruction of its synaptic connectome, and genetic tools for manipulating single, identified neurons allow neural circuit function to be investigated with relative ease and precision. We focus on one of the most complex neurons in the brain of the larva (of either sex), the GABAergic anterior paired lateral neuron (APL). Using behavioral and connectomic analyses, optogenetics, Ca2+ imaging, and pharmacology, we study how APL affects associative olfactory memory. We first provide a detailed account of the structure, regional polarity, connectivity, and metamorphic development of APL, and further confirm that optogenetic activation of APL has an inhibiting effect on its main targets, the mushroom body Kenyon cells. All these findings are consistent with the previously identified function of APL in the sparsening of sensory representations. To our surprise, however, we found that optogenetically activating APL can also have a strong rewarding effect. Specifically, APL activation together with odor presentation establishes an odor-specific, appetitive, associative short-term memory, whereas naive olfactory behavior remains unaffected. An acute, systemic inhibition of dopamine synthesis as well as an ablation of the dopaminergic pPAM neurons impair reward learning through APL activation. Our findings provide a study case of complex circuit function in a numerically simple brain, and suggest a previously unrecognized capacity of central-brain GABAergic neurons to engage in dopaminergic reinforcement.SIGNIFICANCE STATEMENT The single, identified giant anterior paired lateral (APL) neuron is one of the most complex neurons in the insect brain. It is GABAergic and contributes to the sparsening of neuronal activity in the mushroom body, the memory center of insects. We provide the most detailed account yet of the structure of APL in larval Drosophila as a neurogenetically accessible study case. We further reveal that, contrary to expectations, the experimental activation of APL can exert a rewarding effect, likely via dopaminergic reward pathways. The present study both provides an example of unexpected circuit complexity in a numerically simple brain, and reports an unexpected effect of activity in central-brain GABAergic circuits.

Optogenetics: Illuminating neuronal circuits of memory formation.

Optogenetics has revolutionized the field of neuroscience. Within the last decades the development and use of optogenetics gained enormous importance for the identification of functional synaptic connections. Employing optogenetic tools in anatomically defined pathways offers a straightforward strategy to demonstrate neuronal sufficiency, even during state-dependent activity within a neuronal network. Hunger, thirst, fatigue or motivation each impact an animal's behavior and determine the internal states that tune neuronal pathways to generate context-appropriate actions. In particular, higher order brain processes, such as learning and memory formation, are often state-dependent and here optogenetics can provide the means to identify and investigate the neuronal pathways involved. Our aim with this article is to focus on the possibilities and limitations of optogenetic tools for dissecting the neuronal circuits underlying learning and memory formation in Drosophila, while emphasizing what these approaches can tell us about neuronal circuit function in general.

Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate.

Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well-characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE), a key enzyme for mammalian peptide processing. By combining peptidomics and neurogenetics, we addressed the role of carboxypeptidase D (dCPD) in global neuropeptide processing and selected peptide-regulated behaviours in Drosophila. We found that a deficiency in dCPD results in C-terminally extended peptides across the peptidome, suggesting that dCPD took over CPE function in the fruit fly. dCPD is widely expressed throughout the nervous system, including peptidergic neurons in the mushroom body and neuroendocrine cells expressing adipokinetic hormone. Conditional hypomorphic mutation in the dCPD-encoding gene silver in the larva causes lethality, and leads to deficits in starvation-induced hyperactivity and appetitive gustatory preference, as well as to reduced viability and activity levels in adults. A phylogenomic analysis suggests that loss of CPE is not common to insects, but only occurred in Hymenoptera and Diptera. Our results show that dCPD is a key enzyme for neuropeptide processing and peptide-regulated behaviour in Drosophila. dCPD thus appears as a suitable target to genetically shut down total neuropeptide production in peptidergic neurons. The persistent occurrence of CPD in insect genomes may point to important further CPD functions beyond neuropeptide processing which cannot be fulfilled by CPE.

Photoreceptors for immediate effects of light on circadian behavior.

Animals need to sharpen their behavioral output in order to adapt to a variable environment. Hereby, light is one of the most pivotal environmental signals and thus behavioral plasticity in response to light can be observed in diurnal animals, including humans. Furthermore, light is the main entraining signal of the clock, yet immediate effects of light enhance or overwrite circadian output and thereby mask circadian behavior. In Drosophila, such masking effects are most evident as a lights-on response in two behavioral rhythms - the emergence of the adult insect from the pupa, called eclosion, and the diurnal rhythm of locomotor activity. Here, we show that the immediate effect of light on eclosion depends solely on R8 photoreceptors of the eyes. In contrast, the increase in activity by light at night is triggered by different cells and organs that seem to compensate for the loss of each other, potentially to ensure behavioral plasticity.

A behavior-based circuit model of how outcome expectations organize learned behavior in larval Drosophila.

Drosophila larvae combine a numerically simple brain, a correspondingly moderate behavioral complexity, and the availability of a rich toolbox for transgenic manipulation. This makes them attractive as a study case when trying to achieve a circuit-level understanding of behavior organization. From a series of behavioral experiments, we suggest a circuitry of chemosensory processing, odor-tastant memory trace formation, and the "decision" process to behaviorally express these memory traces--or not. The model incorporates statements about the neuronal organization of innate vs. conditioned chemosensory behavior, and the types of interaction between olfactory and gustatory pathways during the establishment as well as the behavioral expression of odor-tastant memory traces. It in particular suggests that innate olfactory behavior is responsive in nature, whereas conditioned olfactory behavior is captured better when seen as an action in pursuit of its outcome. It incorporates the available neuroanatomical and behavioral data and thus should be useful as scaffold for the ongoing investigations of the chemo-behavioral system in larval Drosophila.

Dissection of the peripheral motion channel in the visual system of Drosophila melanogaster.

In the eye, visual information is segregated into modalities such as color and motion, these being transferred to the central brain through separate channels. Here, we genetically dissect the achromatic motion channel in the fly Drosophila melanogaster at the level of the first relay station in the brain, the lamina, where it is split into four parallel pathways (L1-L3, amc/T1). The functional relevance of this divergence is little understood. We now show that the two most prominent pathways, L1 and L2, together are necessary and largely sufficient for motion-dependent behavior. At high pattern contrast, the two pathways are redundant. At intermediate contrast, they mediate motion stimuli of opposite polarity, L2 front-to-back, L1 back-to-front motion. At low contrast, L1 and L2 depend upon each other for motion processing. Of the two minor pathways, amc/T1 specifically enhances the L1 pathway at intermediate contrast. L3 appears not to contribute to motion but to orientation behavior.

Drosophila larvae establish appetitive olfactory memories via mushroom body neurons of embryonic origin.

Insect mushroom bodies are required for diverse behavioral functions, including odor learning and memory. Using the numerically simple olfactory pathway of the Drosophila melanogaster larva, we provide evidence that the formation of appetitive olfactory associations relies on embryonic-born intrinsic mushroom body neurons (Kenyon cells). The participation of larval-born Kenyon cells, i.e., neurons that become gradually integrated in the developing mushroom body during larval life, in this task is unlikely. These data provide important insights into how a small set of identified Kenyon cells can store and integrate olfactory information in a developing brain. To investigate possible functional subdivisions of the larval mushroom body, we anatomically disentangle its input and output neurons at the single-cell level. Based on this approach, we define 10 subdomains of the larval mushroom body that may be implicated in mediating specific interactions between the olfactory pathway, modulatory neurons, and neuronal output.

Endocrine signals fine-tune daily activity patterns in Drosophila.

Animals need to balance competitive behaviors to maintain internal homeostasis. The underlying mechanisms are complex but typically involve neuroendocrine signaling. Using Drosophila, we systematically manipulated signaling between energy-mobilizing endocrine cells producing adipokinetic hormone (AKH), octopaminergic neurons, and the energy-storing fat body to assess whether this neuroendocrine axis involved in starvation-induced hyperactivity also balances activity levels under ad libitum access to food. Our results suggest that AKH signals via two divergent pathways that are mutually competitive in terms of activity and rest. AKH increases activity via the octopaminergic system during the day, while it prevents high activity levels during the night by signaling to the fat body. This regulation involves feedback signaling from octopaminergic neurons to AKH-producing cells (APCs). APCs are known to integrate a multitude of metabolic and endocrine signals. Our results add a new facet to the versatile regulatory functions of APCs by showing that their output contributes to shape the daily activity pattern under ad libitum access to food.

Electric shock-induced associative olfactory learning in Drosophila larvae.

Associative plasticity is a basic essential attribute of nervous systems. As shown by numerous reports, Drosophila is able to establish simple forms of appetitive and aversive olfactory associations at both larval and adult stages. Whereas most adult studies on aversive learning employed electric shock as a negative reinforcer, larval paradigms essentially utilized gustatory stimuli to create negative associations, a discrepancy that limits the comparison of data. To overcome this drawback, we critically revisited larval odor-electric shock conditioning. First, we show that lithium chloride (LiCl), which was used in all previous larval electric shock paradigms, is not required per se in larval odor-electric shock learning. This is of considerable practical advantage because beside its peculiar effects LiCl is attractive to larvae at low concentration that renders comparative learning studies on genetically manipulated larvae complicated. Second, we confirm that in both a 2-odor reciprocal and a 1-odor nonreciprocal conditioning regimen, larvae are able to associate an odor with electric shock. In the latter experiments, initial learning scores reach an asymptote after 5 training trials, and aversive memory is still detectable after 60 min. Our experiments provide a comprehensive basis for future comparisons of larval olfactory conditioning reinforced by different modalities, for studies aimed at analyzing odor-electric shock learning in the larva and the adult, and for investigations of the cellular and molecular substrate of aversive olfactory learning in the simple Drosophila model.

A Novel Thermal-Visual Place Learning Paradigm for Honeybees (Apis mellifera).

Honeybees (Apis mellifera) have fascinating navigational skills and learning capabilities in the field. To decipher the mechanisms underlying place learning in honeybees, we need paradigms to study place learning of individual honeybees under controlled laboratory conditions. Here, we present a novel visual place learning arena for honeybees which relies on high temperatures as aversive stimuli. Honeybees learn to locate a safe spot in an unpleasantly warm arena, relying on a visual panorama. Bees can solve this task at a temperature of 46°C, while at temperatures above 48°C bees die quickly. This new paradigm, which is based on pioneering work on Drosophila, allows us now to investigate thermal-visual place learning of individual honeybees in the laboratory, for example after controlled genetic knockout or pharmacological intervention.

Antinociceptive modulation by the adhesion GPCR CIRL promotes mechanosensory signal discrimination.

Adhesion-type GPCRs (aGPCRs) participate in a vast range of physiological processes. Their frequent association with mechanosensitive functions suggests that processing of mechanical stimuli may be a common feature of this receptor family. Previously, we reported that the Drosophila aGPCR CIRL sensitizes sensory responses to gentle touch and sound by amplifying signal transduction in low-threshold mechanoreceptors (Scholz et al., 2017). Here, we show that Cirl is also expressed in high-threshold mechanical nociceptors where it adjusts nocifensive behaviour under physiological and pathological conditions. Optogenetic in vivo experiments indicate that CIRL lowers cAMP levels in both mechanosensory submodalities. However, contrasting its role in touch-sensitive neurons, CIRL dampens the response of nociceptors to mechanical stimulation. Consistent with this finding, rat nociceptors display decreased Cirl1 expression during allodynia. Thus, cAMP-downregulation by CIRL exerts opposing effects on low-threshold mechanosensors and high-threshold nociceptors. This intriguing bipolar action facilitates the separation of mechanosensory signals carrying different physiological information.

Linking physiological processes and feeding behaviors by octopamine.

The biogenic amine octopamine and to some extent its precursor tyramine function as an alerting signal in insects. Octopaminergic/tyraminergic neurons arborize in most parts of the central nervous system and additionally reach almost all peripheral organs, tissues, and muscles. Indeed, octopamine is involved in motivation, arousal, and the initiation of different behaviors reflecting its function as an alerting signal. A well-studied example of octopamine function is feeding behavior in Drosophila. Here, the amine is involved in food search, sugar/bitter sensitivity, food intake, and starvation-induced hyperactivity. Thereby octopamine modulates feeding initiation in response to internal needs and external stimuli. Additionally, it seems that octopamine/tyramine orchestrate behaviors such as locomotion and feeding or flight and song production to adapt the behavioral outcome of an animal to physiological and environmental conditions. There is a possibility that octopamine and tyramine are required in the selection of behaviors in insects.

Neuropeptides in modulation of Drosophila behavior: how to get a grip on their pleiotropic actions.

Neuropeptides constitute a large and diverse class of signaling molecules that are produced by many types of neurons, neurosecretory cells, endocrines and other cells. Many neuropeptides display pleiotropic actions either as neuromodulators, co-transmitters or circulating hormones, while some play these roles concurrently. Here, we highlight pleiotropic functions of neuropeptides and different levels of neuropeptide signaling in the brain, from context-dependent orchestrating signaling by higher order neurons, to local executive modulation in specific circuits. Additionally, orchestrating neurons receive peptidergic signals from neurons conveying organismal internal state cues and relay these to executive circuits. We exemplify these levels of signaling with four neuropeptides, SIFamide, short neuropeptide F, allatostatin-A and leucokinin, each with a specific expression pattern and level of complexity in signaling.

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 we provide evidence 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, we argue that dopaminergic pPAM neurons mediate reward information to the Kenyon cells, and in turn receive feedback from Kenyon cells. We further show 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. Our data suggest that recurrent signaling routes within the larval mushroom body circuitry may represent a mechanism subserving memory stabilization.

Odor-taste learning in Drosophila larvae.

The Drosophila larva is an attractive model system to study fundamental questions in the field of neuroscience. Like the adult fly, the larva offers a seemingly unlimited genetic toolbox, which allows one to visualize, silence or activate neurons down to the single cell level. This, combined with its simplicity in terms of cell numbers, offers a useful system to study the neuronal correlates of complex processes including associative odor-taste learning and memory formation. Here, we summarize the current knowledge about odor-taste learning and memory at the behavioral level and integrate the recent progress on the larval connectome to shed light on the sub-circuits that allow Drosophila larvae to integrate present sensory input in the context of past experience and to elicit an appropriate behavioral response.

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. We here describe 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.

Synthetic Light-Activated Ion Channels for Optogenetic Activation and Inhibition.

Optogenetic manipulation of cells or living organisms became widely used in neuroscience following the introduction of the light-gated ion channel channelrhodopsin-2 (ChR2). ChR2 is a non-selective cation channel, ideally suited to depolarize and evoke action potentials in neurons. However, its calcium (Ca2+) permeability and single channel conductance are low and for some applications longer-lasting increases in intracellular Ca2+ might be desirable. Moreover, there is need for an efficient light-gated potassium (K+) channel that can rapidly inhibit spiking in targeted neurons. Considering the importance of Ca2+ and K+ in cell physiology, light-activated Ca2+-permeant and K+-specific channels would be welcome additions to the optogenetic toolbox. Here we describe the engineering of novel light-gated Ca2+-permeant and K+-specific channels by fusing a bacterial photoactivated adenylyl cyclase to cyclic nucleotide-gated channels with high permeability for Ca2+ or for K+, respectively. Optimized fusion constructs showed strong light-gated conductance in Xenopus laevis oocytes and in rat hippocampal neurons. These constructs could also be used to control the motility of Drosophila melanogaster larvae, when expressed in motoneurons. Illumination led to body contraction when motoneurons expressed the light-sensitive Ca2+-permeant channel, and to body extension when expressing the light-sensitive K+ channel, both effectively and reversibly paralyzing the larvae. Further optimization of these constructs will be required for application in adult flies since both constructs led to eclosion failure when expressed in motoneurons.

SIFamide Translates Hunger Signals into Appetitive and Feeding Behavior in Drosophila.

Animal behavior is, on the one hand, controlled by neuronal circuits that integrate external sensory stimuli and induce appropriate motor responses. On the other hand, stimulus-evoked or internally generated behavior can be influenced by motivational conditions, e.g., the metabolic state. Motivational states are determined by physiological parameters whose homeostatic imbalances are signaled to and processed within the brain, often mediated by modulatory peptides. Here, we investigate the regulation of appetitive and feeding behavior in the fruit fly, Drosophila melanogaster. We report that four neurons in the fly brain that release SIFamide are integral elements of a complex neuropeptide network that regulates feeding. We show that SIFamidergic cells integrate feeding stimulating (orexigenic) and feeding suppressant (anorexigenic) signals to appropriately sensitize sensory circuits, promote appetitive behavior, and enhance food intake. Our study advances the cellular dissection of evolutionarily conserved signaling pathways that convert peripheral metabolic signals into feeding-related behavior.

Potency of transgenic effectors for neurogenetic manipulation in Drosophila larvae.

Genetic manipulations of neuronal activity are a cornerstone of studies aimed to identify the functional impact of defined neurons for animal behavior. With its small nervous system, rapid life cycle, and genetic amenability, the fruit fly Drosophila melanogaster provides an attractive model system to study neuronal circuit function. In the past two decades, a large repertoire of elegant genetic tools has been developed to manipulate and study neural circuits in the fruit fly. Current techniques allow genetic ablation, constitutive silencing, or hyperactivation of neuronal activity and also include conditional thermogenetic or optogenetic activation or inhibition. As for all genetic techniques, the choice of the proper transgenic tool is essential for behavioral studies. Potency and impact of effectors may vary in distinct neuron types or distinct types of behavior. We here systematically test genetic effectors for their potency to alter the behavior of Drosophila larvae, using two distinct behavioral paradigms: general locomotor activity and directed, visually guided navigation. Our results show largely similar but not equal effects with different effector lines in both assays. Interestingly, differences in the magnitude of induced behavioral alterations between different effector lines remain largely consistent between the two behavioral assays. The observed potencies of the effector lines in aminergic and cholinergic neurons assessed here may help researchers to choose the best-suited genetic tools to dissect neuronal networks underlying the behavior of larval fruit flies.