Drosophila mutants lacking the glial neurotransmitter-modifying enzyme Ebony exhibit low neurotransmitter levels and altered behavior.

Inhibitors of enzymes that inactivate amine neurotransmitters (dopamine, serotonin), such as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO), are thought to increase neurotransmitter levels and are widely used to treat Parkinson's disease and psychiatric disorders, yet the role of these enzymes in regulating behavior remains unclear. Here, we investigated the genetic loss of a similar enzyme in the model organism Drosophila melanogaster. Because the enzyme Ebony modifies and inactivates amine neurotransmitters, its loss is assumed to increase neurotransmitter levels, increasing behaviors such as aggression and courtship and decreasing sleep. Indeed, ebony mutants have been described since 1960 as "aggressive mutants," though this behavior has not been quantified. Using automated machine learning-based analyses, we quantitatively confirmed that ebony mutants exhibited increased aggressive behaviors such as boxing but also decreased courtship behaviors and increased sleep. Through tissue-specific knockdown, we found that ebony's role in these behaviors was specific to glia. Unexpectedly, direct measurement of amine neurotransmitters in ebony brains revealed that their levels were not increased but reduced. Thus, increased aggression is the anomalous behavior for this neurotransmitter profile. We further found that ebony mutants exhibited increased aggression only when fighting each other, not when fighting wild-type controls. Moreover, fights between ebony mutants were less likely to end with a clear winner than fights between controls or fights between ebony mutants and controls. In ebony vs. control fights, ebony mutants were more likely to win. Together, these results suggest that ebony mutants exhibit prolonged aggressive behavior only in a specific context, with an equally dominant opponent.

Sensory integration: Time and temperature regulate fly siesta.

Temperatures outside the preferred range require flies to acutely adjust their behavior. A new study finds that heat-sensing neurons provide input to fly circadian clock neurons to extend the daytime siesta, allowing flies to sleep through excessive daytime heat.

Dietary restriction ameliorates TBI-induced phenotypes in Drosophila melanogaster.

Traumatic brain injury (TBI) affects millions annually and is associated with long-term health decline. TBI also shares molecular and cellular hallmarks with neurodegenerative diseases (NDs), typically increasing in prevalence with age, and is a major risk factor for developing neurodegeneration later in life. While our understanding of genes and pathways that underlie neurotoxicity in specific NDs has advanced, we still lack a complete understanding of early molecular and physiological changes that drive neurodegeneration, particularly as an individual ages following a TBI. Recently Drosophila has been introduced as a model organism for studying closed-head TBI. In this paper, we deliver a TBI to flies early in adult life, and then measure molecular and physiological phenotypes at short-, mid-, and long-term timepoints following the injury. We aim to identify the timing of changes that contribute to neurodegeneration. Here we confirm prior work demonstrating a TBI-induced decline in lifespan, and present evidence of a progressive decline in locomotor function, robust acute and modest chronic neuroinflammation, and a late-onset increase in protein aggregation. We also present evidence of metabolic dysfunction, in the form of starvation sensitivity and decreased lipids, that persists beyond the immediate injury response, but does not differ long-term. An intervention of dietary restriction (DR) partially ameliorates some TBI-induced phenotypes, including lifespan and locomotor function, though it does not alter the pattern of starvation sensitivity of injured flies. In the future, molecular pathways identified as altered following TBI-particularly in the short-, or mid-term-could present potential therapeutic targets.

Circadian regulation of mitochondrial uncoupling and lifespan.

Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target.

A course-based undergraduate research experience examining neurodegeneration in Drosophila melanogaster teaches students to think, communicate, and perform like scientists.

As educators strive to incorporate more active learning and inquiry-driven exercises into STEM curricula, Course-based Undergraduate Research Experiences (CUREs) are becoming more common in undergraduate laboratory courses. Here we detail a CURE developed in an upper-level undergraduate genetics course at Yeshiva University, centered on the Drosophila melanogaster ortholog of the human neurodegeneration locus PLA2G6/PARK14. Drosophila PLA2G6 mutants exhibit symptoms of neurodegeneration, such as attenuated lifespan and decreased climbing ability with age, which can be replicated by neuron-specific knockdown of PLA2G6. To ask whether the neurodegeneration phenotype could be caused by loss of PLA2G6 in specific neuronal subtypes, students used GAL4-UAS to perform RNAi knockdown of PLA2G6 in subsets of neurons in the Drosophila central nervous system and measured age-dependent climbing ability. We organized our learning objectives for the CURE into three broad goals of having students think, communicate, and perform like scientists. To assess how well students achieved these goals, we developed a detailed rubric to analyze written lab reports, administered pre- and post-course surveys, and solicited written feedback. We observed striking gains related to all three learning goals, and students reported a high degree of satisfaction. We also observed significantly improved understanding of the scientific method by students in the CURE as compared to the prior year's non-CURE genetics lab students. Thus, this CURE can serve as a template to successfully engage students in novel research, improve understanding of the scientific process, and expose students to the use of Drosophila as a model for human neurodegenerative disease.

Dissection of central clock function in Drosophila through cell-specific CRISPR-mediated clock gene disruption.

In Drosophila, ~150 neurons expressing molecular clock proteins regulate circadian behavior. Sixteen of these neurons secrete the neuropeptide Pdf and have been called 'master pacemakers' because they are essential for circadian rhythms. A subset of Pdf+ neurons (the morning oscillator) regulates morning activity and communicates with other non-Pdf+ neurons, including a subset called the evening oscillator. It has been assumed that the molecular clock in Pdf+ neurons is required for these functions. To test this, we developed and validated Gal4-UAS based CRISPR tools for cell-specific disruption of key molecular clock components, period and timeless. While loss of the molecular clock in both the morning and evening oscillators eliminates circadian locomotor activity, the molecular clock in either oscillator alone is sufficient to rescue circadian locomotor activity in the absence of the other. This suggests that clock neurons do not act in a hierarchy but as a distributed network to regulate circadian activity.

A Drosophila model of Fragile X syndrome exhibits defects in phagocytosis by innate immune cells.

Fragile X syndrome, the most common known monogenic cause of autism, results from the loss of FMR1, a conserved, ubiquitously expressed RNA-binding protein. Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. We found that Drosophila melanogaster Fmr1 mutants exhibit increased sensitivity to bacterial infection and decreased phagocytosis of bacteria by systemic immune cells. Using tissue-specific RNAi-mediated knockdown, we showed that Fmr1 plays a cell-autonomous role in the phagocytosis of bacteria. Fmr1 mutants also exhibit delays in two processes that require phagocytosis by glial cells, the immune cells in the brain: neuronal clearance after injury in adults and the development of the mushroom body, a brain structure required for learning and memory. Delayed neuronal clearance is associated with reduced recruitment of activated glia to the site of injury. These results suggest a previously unrecognized role for Fmr1 in regulating the activation of phagocytic immune cells both in the body and the brain.

Bitter taste receptors confer diverse functions to neurons.

Bitter compounds elicit an aversive response. In Drosophila, bitter-sensitive taste neurons coexpress many members of the Gr family of taste receptors. However, the molecular logic of bitter signaling is unknown. We used an in vivo expression approach to analyze the logic of bitter taste signaling. Ectopic or overexpression of bitter Grs increased endogenous responses or conferred novel responses. Surprisingly, expression of Grs also suppressed many endogenous bitter responses. Conversely, deletion of an endogenous Gr led to novel responses. Expression of individual Grs conferred strikingly different effects in different neurons. The results support a model in which bitter Grs interact, exhibiting competition, inhibition, or activation. The results have broad implications for the problem of how taste systems evolve to detect new environmental dangers.

Electrophysiological recording from Drosophila labellar taste sensilla.

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.