PUBLICATIONS

  • Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster
    Huda, A., Vaden T. J., Bai H., Rawls R. T., Peppers R. J., Monck C. F., Holley H. D., Castaneda A. N., Ni. L

    JoVE, 2025

    DOI: 10.3791/67964

    Video DOI: 10.3791/67964-v

    Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

  • Functional labeling of individualized postsynaptic neurons using optogenetics and trans-Tango
    Castaneda, A. N., Huda, A., Whitaker, I. B. M., Reily, J. E., Shelby, G. S., Ni, L.

    PLOS Genetics, 2024

    DOI: 10.1371/journal.pgen.1011190

    A population of neurons interconnected by synapses constitutes a neural circuit, which performs specific functions upon activation. It is essential to identify both anatomical and functional entities of neural circuits to comprehend the components and processes necessary for healthy brain function and the changes that characterize brain disorders. To date, few methods are available to study these two aspects of a neural circuit simultaneously. In this study, we developed FLIPSOT, or functional labeling of individualized postsynaptic neurons using optogenetics and trans-Tango. FLIPSOT uses (1) trans-Tango to access postsynaptic neurons genetically, (2) optogenetic approaches to activate (FLIPSOTa) or inhibit (FLIPSOTi) postsynaptic neurons in a random and sparse manner, and (3) fluorescence markers tagged with optogenetic genes to visualize these neurons. Therefore, FLIPSOT allows using a presynaptic driver to identify the behavioral function of individual postsynaptic neurons. It is readily applied to identify functions of individual postsynaptic neurons and has the potential to be adapted for use in mammalian circuits.

  • Using Drosophila Two-Choice Assay to Study Optogenetics in Hands-On Neurobiology Laboratory Activities
    Fu, Z., Huda, A., Kimbrough, I. F., Ni, L.

    Journal of Undergraduate Neuroscience Education, 2024

    DOI: 10.59390/SDWH9124

    Optogenetics has made a significant impact on neuroscience, allowing activation and inhibition of neural activity with exquisite spatiotemporal precision in response to light. In this lab session, we use fruit flies as a model organism to enable students to understand the fundamentals of optogenetics through hands-on activities. The CsChrimson channelrhodopsin, a light-activated cation channel, is expressed in sweet and bitter responsive neurons. Students are required to use two-choice assays to explore the causality between the light activation of these neurons and the appetitive and avoidance behaviors of the fruit flies. To quantify their observations, students need to calculate preference indices and use the student’s t-test to analyze their data.

  • Responses to Temperature Changes of Different Drosophila Species
    Huda, A., Omelchenko, A. A., Vaden, T. J., Castaneda, A. N., Ni, L.

    Journal of Experimental Biology, 2022

    DOI: 10.1242/jeb.243708

    Temperature is a critical environmental variable that affects the distribution, survival and reproduction of most animals. Although temperature receptors have been identified in many animals, how these receptors respond to temperature is still unclear. Here, we describe an automated tracking method for studying the thermotactic behaviors of Drosophila larvae and adults. We built optimal experimental setups to capture behavioral recordings and analyzed them using free software, Fiji and TrackMate, which do not require programming knowledge. Then, we applied the adult thermotactic two-choice assay to examine the movement and temperature preferences of nine Drosophila species. The ability or inclination to move varied among these species and at different temperatures. Distinct species preferred various ranges of temperatures. Wild-type D. melanogaster flies avoided the warmer temperature in the warm avoidance assay and the cooler temperature in the cool avoidance assay. Conversely, D. bipectinata and D. yakuba did not avoid warm or cool temperatures in the respective assays, and D. biarmipes and D. mojavensis did not avoid the warm temperature in the warm avoidance assay. These results demonstrate that Drosophila species have different mobilities and temperature preferences, which will benefit further research in exploring molecular mechanisms of temperature responsiveness.

  • Gut bacteria regulate the pathogenesis of Huntington’s disease in Drosophila
    Chongtham, A., Yoo, J. H., Chin, T. M., Akingbesot, N. D., Huda, A, Khoshnan, A., Dickinson, M.

    Journal of Neurobiology of Disease, 2022

    DOI: 10.3389/fnins.2022.902205

    Changes in the composition of gut microbiota are implicated in the pathogenesis of several neurodegenerative disorders. Here, we investigated whether gut bacteria affect the progression of Huntington’s disease (HD) in transgenic Drosophila melanogaster (fruit fly) models expressing full-length or N-terminal fragments of human mutant huntingtin (HTT) protein. We find that elimination of commensal gut bacteria by antibiotics reduces the aggregation of amyloidogenic N-terminal fragments of HTT and delays the development of motor defects. Conversely, colonization of HD flies with Escherichia coli (E. coli), a known pathobiont of human gut with links to neurodegeneration and other morbidities, accelerates HTT aggregation, aggravates immobility, and shortens lifespan. Similar to antibiotics, treatment of HD flies with small compounds such as luteolin, a flavone, or crocin a beta-carotenoid, ameliorates disease phenotypes, and promotes survival. Crocin prevents colonization of E. colii in the gut and alters the levels of commensal bacteria, which may be linked to its protective effects. The opposing effects of E. coli and crocin on HTT aggregation, motor defects, and survival in transgenic Drosophila models support the involvement of gut-brain networks in the pathogenesis of HD.

  • Flies Regulate Wing Motion via Active Control of a Dual-Function Gyroscope
    Dickerson, B. H., de Souza, A. M., Huda, A, Dickinson, M.

    Current Biology, 2019

    DOI: 10.1016/j.cub.2019.08.065

    Flies execute their remarkable aerial maneuvers using a set of wing steering muscles, which are activated at specific phases of the stroke cycle. The activation phase of these muscles—which determines their biomechanical output—arises via feedback from mechanoreceptors at the base of the wings and structures unique to flies called halteres. Evolved from the hindwings, the tiny halteres oscillate at the same frequency as the wings, although they serve no aerodynamic function and are thought to act as gyroscopes. Like the wings, halteres possess minute control muscles whose activity is modified by descending visual input, raising the possibility that flies control wing motion by adjusting the motor output of their halteres, although this hypothesis has never been directly tested. Here, using genetic techniques possible in Drosophila melanogaster, we tested the hypothesis that visual input during flight modulates haltere muscle activity and that this, in turn, alters the mechanosensory feedback that regulates the wing steering muscles. Our results suggest that rather than acting solely as a gyroscope to detect body rotation, halteres also function as an adjustable clock to set the spike timing of wing motor neurons, a specialized capability that evolved from the generic flight circuitry of their four-winged ancestors. In addition to demonstrating how the efferent control loop of a sensory structure regulates wing motion, our results provide insight into the selective scenario that gave rise to the evolution of halteres.

  • Distinct activity-gated pathways mediate attraction and aversion to CO2 in Drosophila
    Van Breugel, F., Huda, A, Dickinson, M. H.

    Nature, 2018

    DOI: 10.1038/s41586-018-0732-8

    Carbon dioxide is produced by many organic processes and is a convenient volatile cue for insects1 that are searching for blood hosts, flowers, communal nests, fruit and wildfires. Although Drosophila melanogaster feed on yeast that produce CO2 and ethanol during fermentation, laboratory experiments suggest that walking flies avoid CO2. Here we resolve this paradox by showing that both flying and walking Drosophila find CO2 attractive, but only when they are in an active state associated with foraging. Their aversion to CO2 at low-activity levels may be an adaptation to avoid parasites that seek CO2, or to avoid succumbing to respiratory acidosis in the presence of high concentrations of CO2 that exist in nature. In contrast to CO2, flies are attracted to ethanol in all behavioural states, and invest twice the time searching near ethanol compared to CO2. These behavioural differences reflect the fact that ethanol is a unique signature of yeast fermentation, whereas CO2 is generated by many natural processes. Using genetic tools, we determined that the evolutionarily conserved ionotropic co-receptor IR25a is required for CO2 attraction, and that the receptors necessary for CO2 avoidance are not involved in this attraction. Our study lays the foundation for future research to determine the neural circuits that underlie both state- and odorant-dependent decision-making in Drosophila.

  • An Array of Descending Visual Interneurons Encoding Self-Motion in Drosophila
    Suver, M., Huda, A, Iwasaki, N., Safarik, S., Dickinson, M. H.

    Journal of Neuroscience, 2016

    DOI: 10.1523/JNEUROSCI.2277-16.2016

    The means by which brains transform sensory information into coherent motor actions is poorly understood. In flies, a relatively small set of descending interneurons are responsible for conveying sensory information and higher-order commands from the brain to motor circuits in the ventral nerve cord. Here, we describe three pairs of genetically identified descending interneurons that integrate information from wide-field visual interneurons and project directly to motor centers controlling flight behavior. We measured the physiological responses of these three cells during flight and found that they respond maximally to visual movement corresponding to rotation around three distinct body axes. After characterizing the tuning properties of an array of nine putative upstream visual interneurons, we show that simple linear combinations of their outputs can predict the responses of the three descending cells. Last, we developed a machine vision-tracking system that allows us to monitor multiple motor systems simultaneously and found that each visual descending interneuron class is correlated with a discrete set of motor programs.