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.     

Identification of neurons responsible for feeding behavior in the Drosophila brain

Drosophila melanogaster feeds mainly on rotten fruits,which contain many kinds of sugar.Thus,the sense of sweet taste has evolved to serve as a dominant regulator and driver of feeding behavior.Although several sugar receptors have been described,it remains poorly understood how the sensory input is transformed into an appetitive behavior.Here,we used a neural silencing approach to screen brain circuits,and identified neurons labeled by three Gal4 lines that modulate Drosophila feeding behavior.These three Gal4 lines labeled neurons mainly in the suboesophageal ganglia(SOG),which is considered to be the fly’s primary taste center.When we blocked the activity of these neurons,flies decreased their sugar consumption significantly.In contrast,activation of these neurons resulted in enhanced feeding behavior and increased food consumption not only towards sugar,but to an array of food sources.Moreover,upon neuronal activation,the flies demonstrated feeding behavior even in the absence of food,which suggests that neuronal activation can replace food as a stimulus for feeding behavior.These findings indicate that these Gal4-labeled neurons,which function downstream of sensory neurons and regulate feeding behavior towards different food sources is necessary in Drosophila feeding control.     

Allatostatin A Signalling in Drosophila Regulates Feeding and Sleep and Is Modulated by PDF

Feeding and sleep are fundamental behaviours with significant interconnections and cross-modulations. The circadian system and peptidergic signals are important components of this modulation, but still little is known about the mechanisms and networks by which they interact to regulate feeding and sleep. We show that specific thermogenetic activation of peptidergic Allatostatin A (AstA)-expressing PLP neurons and enteroendocrine cells reduces feeding and promotes sleep in the fruit fly Drosophila. The effects of AstA cell activation are mediated by AstA peptides with receptors homolog to galanin receptors subserving similar and apparently conserved functions in vertebrates. We further identify the PLP neurons as a downstream target of the neuropeptide pigment-dispersing factor (PDF), an output factor of the circadian clock. PLP neurons are contacted by PDF-expressing clock neurons, and express a functional PDF receptor demonstrated by cAMP imaging. Silencing of AstA signalling and continuous input to AstA cells by tethered PDF changes the sleep/activity ratio in opposite directions but does not affect rhythmicity. Taken together, our results suggest that pleiotropic AstA signalling by a distinct neuronal and enteroendocrine AstA cell subset adapts the fly to a digestive energy-saving state which can be modulated by PDF.     

Dopamine Dynamics and Signaling in Drosophila: An Overview of Genes,Drugs and Behavioral Paradigms

Changes in dopamine (DA) signaling have been implicated in a number of human neurologic and psychiatric disorders. Similarly, defects in DA signaling in the fruit fly, Drosophila melanogaster, have also been associated with several behavioral defects. As most genes involved in DA synthesis, transport, secretion, and signaling are conserved between species, Drosophila is a powerful genetic model organism to study the regulation of DA signaling in vivo. In this review, we will provide an overview of the genes and drugs that regulate DA biology in Drosophila. Furthermore, we will discuss the behavioral paradigms that are regulated by DA signaling in flies. By analyzing the genes and neuronal circuits that govern such behaviors using sophisticated genetic, pharmacologic, electrophysiologic, and imaging approaches in Drosophila, we will likely gain a better understanding about how this neuromodulator regulates motor tasks and cognition in humans.     

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms^1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night ³. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons³. Drosophila locomotor activity is a well known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms⁴. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR³. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies^5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.     

Neuronal necrosis and spreading death in a Drosophila genetic model

Brain ischemia often results in neuronal necrosis, which may spread death to neighboring cells. However, the molecular events of neuronal necrosis and the mechanisms of this spreading death are poorly understood due to the limited genetic tools available for deciphering complicated responses in mammalian brains. Here, we engineered a Drosophila model of necrosis in a sub-population of neurons by expressing a leaky cation channel in the Drosophila eye. Expression of this channel caused necrosis in defined neurons as well as extensive spreading of cell death. Jun N-terminal kinase (JNK)-mediated, caspase-independent apoptosis was the primary mechanism of cell death in neurons, while caspase-dependent apoptosis was primarily involved in non-neuronal cell death. Furthermore, the JNK activation in surrounding neurons was triggered by reactive oxygen species (ROS) and Eiger (Drosophila tumor necrosis factor α (TNFα)) released from necrotic neurons. Because the Eiger/ROS/JNK signaling was also required for cell death induced by hypoxia and oxidative stress, our fly model of spreading death may be similar to brain ischemia in mammals. We performed large-scale genetic screens to search for novel genes functioning in necrosis and/or spreading death, from which we identified several classes of genes. Among them, Rho-associated kinase (ROCK) had been reported as a promising drug target for stroke treatment with undefined mechanisms. Our data indicate that ROCK and the related trafficking pathway genes regulate neuronal necrosis. We propose the suppression of the function of the trafficking system, ROS and cytokines, such as TNFα, as translational applications targeting necrosis and spreading death.     

A novel satiety sensor detects circulating glucose and suppresses food consumption via insulin-producing cells in Drosophila

Sensing satiety is a crucial survival skill for all animal species including human. Despite the discovery of numerous neuromodulators that regulate food intake in Drosophila, the mechanism of satiety sensing remains largely elusive. Here, we investigated how neuropeptidergic circuitry conveyed satiety state to influence flies’ food consumption. Drosophila tackykinin (DTK) and its receptor TAKR99D were identified in an RNAi screening as feeding suppressors. Two pairs of DTK⁺ neurons in the fly brain could be activated by elevated D-glucose in the hemolymph and imposed a suppressive effect on feeding. These DTK⁺ neurons formed a two-synapse circuitry targeting insulin-producing cells, a well-known feeding suppressor, via TAKR99D⁺ neurons, and this circuitry could be rapidly activated during food ingestion and cease feeding. Taken together, we identified a novel satiety sensor in the fly brain that could detect specific circulating nutrients and in turn modulate feeding, shedding light on the neural regulation of energy homeostasis.Subject terms: Calcium signalling, Nutrient signalling     

Immunohistochemical analysis of a novel dehydroepiandrosterone sulfotransferase-like protein in Drosophila neural circuits

Sulfotransferase (ST)-catalyzed sulfation plays an important role in various neuronal functions such as homeostasis of catecholamine neurotransmitters and hormones. Drosophila is a popular model for the study of memory and behavioral manifestations because it is able to mimic the intricate neuroregulation and recognition in humans. However, there has been no evidence indicating that cytosolic ST(s) is(are) present in Drosophila. The aim of this study is to investigate whether or not cytosolic ST(s) is(are) expressed in the Drosophila nervous system. Immunoblot analysis demonstrated the presence of dehydroepiandrosterone (DHEA) ST-like protein in Drosophila brain and a sensitive fluorometric assay revealed its sulfating activity toward DHEA. Immunohistochemical staining demonstrated this DHEA ST-like protein to be abundant in specific neurons as well as in several bundles of nerve fibers in Drosophila. Clarification of a possible link between ST and a neurotransmitter-mediated effect may eventually aid in designing approaches for alleviating neuronal disorders in humans.     

Ex vivo Culturing of Whole,Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab¹ has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)², was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine³ causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly⁴, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern⁵. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches⁶, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B1⁷, and that is dependent on the activity of the ubiquitin-proteasome system⁶. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.     

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits^1-6. Application of optogenetics^7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways^9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system^14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin^16,17 or halorhodopsin^18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.     

Light activates output from evening neurons and inhibits output from morning neurons in the Drosophila circadian clock

Animal circadian clocks are based on multiple oscillators whose interactions allow the daily control of complex behaviors. The Drosophila brain contains a circadian clock that controls rest–activity rhythms and relies upon different groups of PERIOD (PER)–expressing neurons. Two distinct oscillators have been functionally characterized under light-dark cycles. Lateral neurons (LNs) that express the pigment-dispersing factor (PDF) drive morning activity, whereas PDF-negative LNs are required for the evening activity. In constant darkness, several lines of evidence indicate that the LN morning oscillator (LN-MO) drives the activity rhythms, whereas the LN evening oscillator (LN-EO) does not. Since mutants devoid of functional CRYPTOCHROME (CRY), as opposed to wild-type flies, are rhythmic in constant light, we analyzed transgenic flies expressing PER or CRY in the LN-MO or LN-EO. We show that, under constant light conditions and reduced CRY function, the LN evening oscillator drives robust activity rhythms, whereas the LN morning oscillator does not. Remarkably, light acts by inhibiting the LN-MO behavioral output and activating the LN-EO behavioral output. Finally, we show that PDF signaling is not required for robust activity rhythms in constant light as opposed to its requirement in constant darkness, further supporting the minor contribution of the morning cells to the behavior in the presence of light. We therefore propose that day–night cycles alternatively activate behavioral outputs of the Drosophila evening and morning lateral neurons.     

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.     

Blockade of the release of the neuropeptide leucokinin to determine its possible functions in fly behavior: Chemoreception assays

Previous studies have revealed leucokinin (LK) expression in the brain and ventral ganglion of Drosophila CNS. One pair of protocerebrum neurons located in the lateral horn area (LHLK) surrounds the peduncles of the mushroom bodies while two pairs of subesophageal neurons (SELKs) project extended processes to the tritocerebrum and through a cervical connection to the ventral ganglion. There, axons of eight or nine pairs of abdominal (ABLK) neurons leave the CNS through the abdominal nerves and processes connecting each other ipsilaterally and contralaterally. The neural functions of LK remain largely unknown, especially those related to Drosophila behavior. Here, we have studied the role of LK in olfactory and gustatory perception by keeping the LK neurons electrically silent through targeted expression of inward rectifier K⁺ channels. In order to examine the effects of LK failure, we first analyzed the dehydration response, comparing the leucokinin-silent individuals with their parents as a control. Our results showed significant differences that demonstrate the effectiveness of the method. We then tested the olfactory behavioral response to a set of odorants over a range of concentrations in a T-maze paradigm in which flies were allowed to choose between the odorant and solvent compartments. The feeding preference assays were carried out on microplates in which flies were allowed to choose between two colored tastes. Our results show that the blockade of LK release alters both olfactory and gustatory responses, and are therefore evidence that this neuropeptide also modulates chemosensory responses through LHLK and SELK neurons.     

Fruit fly behavior in response to chemosensory signals

An important question in contemporary sensory neuroscience is how animals perceive their environment and make appropriate behavioral choices based on chemical perceptions. The fruit fly Drosophila melanogaster exhibits robust tastant and odor-evoked behaviors. Understanding how the gustatory and olfactory systems support the perception of these contact and volatile chemicals and translate them into appropriate attraction or avoidance behaviors has made an unprecedented contribution to our knowledge of the organization of chemosensory systems. In this review, I begin by describing the receptors and signaling mechanisms of the Drosophila gustatory and olfactory systems and then highlight their involvement in the control of simple and complex behaviors. The topics addressed include feeding behavior, learning and memory, navigation behavior, neuropeptide modulation of chemosensory behavior, and I conclude with a discussion of recent work that provides insight into pheromone signaling pathways.     

Behavioural Pharmacology in Classical Conditioning of the Proboscis Extension Response in Honeybees (Apis mellifera)

Honeybees (Apis mellifera) are well known for their communication and orientation skills and for their impressive learning capability^1,2. Because the survival of a honeybee colony depends on the exploitation of food sources, forager bees learn and memorize variable flower sites as well as their profitability. Forager bees can be easily trained in natural settings where they forage at a feeding site and learn the related signals such as odor or color. Appetitive associative learning can also be studied under controlled conditions in the laboratory by conditioning the proboscis extension response (PER) of individually harnessed honeybees^3,4. This learning paradigm enables the study of the neuronal and molecular mechanisms that underlie learning and memory formation in a simple and highly reliable way^5-12. A behavioral pharmacology approach is used to study molecular mechanisms. Drugs are injected systemically to interfere with the function of specific molecules during or after learning and memory formation^13-16.Here we demonstrate how to train harnessed honeybees in PER conditioning and how to apply drugs systemically by injection into the bee flight muscle.     

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at ≥6 months after birth. We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.     

Behavioral and electrophysiological analysis of general anesthesia in 3 background strains of Drosophila melanogaster

General anesthetics achieve behavioral unresponsiveness via a mechanism that is incompletely understood. The study of genetic model systems such as the fruit fly Drosophila melanogaster is crucial to advancing our understanding of how anesthetic drugs render animals unresponsive. Previous studies have shown that wild-type control strains differ significantly in their sensitivity to general anesthetics, which potentially introduces confounding factors for comparing genetic mutations placed on these wild-type backgrounds. Here, we examined a variety of behavioral and electrophysiological endpoints in Drosophila, in both adult and larval animals. We characterized these endpoints in 3 commonly used fly strains: wild-type Canton Special (CS), and 2 commonly used white-eyed strains, isoCJ1 and w¹¹¹⁸. We found that CS and isoCJ1 show remarkably similar sensitivity to isoflurane across a variety of behavioral and electrophysiological endpoints. In contrast, w¹¹¹⁸ is resistant to isoflurane compared to the other 2 strains at both the adult and larval stages. This resistance is however not reflected at the level of neurotransmitter release at the larval neuromuscular junction (NMJ). This suggests that the w¹¹¹⁸ strain harbors another mutation that produces isoflurane resistance, by acting on an arousal pathway that is most likely preserved between larval and adult brains. This mutation probably also affects sleep, as marked differences between isoCJ1 and w¹¹¹⁸ have also recently been found for behavioral responsiveness and sleep intensity measures.     

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.