The authors have nothing to disclose.

1. Assembly of the Strike Device (Figure 1)
<ol>
	<li>Remove the plunger from a 1&#160;mL tuberculin syringe. Cut the barrel at the 1&#160;mL mark.</li>
	<li>Remove an aerosol barrier (3 mm height x 4 mm diameter) from a 200&#160;&#181;L pipette tip and use it as an impactor. Place the impactor inside the syringe barrel. Gently tap the barrel to move the impactor to the tip end, with the flat side covering the nozzle opening.</li>
	<li>Attach the barrel tip end to plastic tubing that is connected to the carbon dioxide (CO2) flow regulator of a Drosophila anesthesia station.</li>
	<li>Hold the barrel vertically and clamp it to a standard clamp holder stand so that the impactor stays at the bottom of the barrel.</li>
	<li>Modify a 200&#160;&#181;L pipette tip to make the fly holder.
	<ol>
		<li>Cut 4 mm from the tip to make a 0.8 mm-diameter opening, allowing only the fly head to be exposed. 
		NOTE: The thorax and all the other parts of the fly body will stay inside the pipette tip.</li>
	</ol>
	</li>
	<li>Modify a 1,000&#160;&#181;L pipette tip and a 1&#160;mL syringe needle cap to make the connector.
	<ol>
		<li>Cut off 44 mm from the opening of the tip. Take a 6&#160;mm length of a 1&#160;mL syringe needle cap and push it tightly into the remaining segment of the 1,000&#160;&#181;L pipette tip.</li>
	</ol>
	</li>
</ol>
2. Operation of the Strike Device
<ol>
	<li>Anesthetize a single 2-day-old adult female fly using CO2 on a fly pad.</li>
	<li>Gently transfer it to the fly holder using a fine brush. Tap the holder gently so that the fly head is seen outside of the tip end. If the fly proboscis is exposed outside the tip, gently tuck it back inside the tip with a blunted 1&#160;mL syringe needle. 
	NOTE: Make sure to keep the fly proboscis inside the holder. Otherwise, the fly may die from a sucking proboscis injury.</li>
	<li>Tighten the fly holder to the syringe barrel with the connector so that the fly head is facing downwards.</li>
	<li>Set the gas pressure at 100 kPa. Adjust the flow rate according to the experiment design.</li>
	<li>Quickly turn the flow regulator toggle switch on and off so that the impactor strikes the fly head once.</li>
	<li>Lift the fly holder and move it over a fly pad. Reverse the fly holder and gently tap the side to let the fly out. Leave the fly in an empty vial to recover.</li>
</ol>
3. Video-assisted Movement Tracking
<ol>
	<li>Fill a 6-cm-diameter Petri dish with transparent silicon elastomer to make the tracking arena. Leave a 3&#160;mm space between the silicon and the dish lid to allow the flies to walk freely but not fly.</li>
	<li>Anesthetize four flies from either the sham or treated group each time and place them in the arena. Leave the flies at 22 &#176;C for 1 h.</li>
	<li>Position a Charge-Coupled Device (CCD) camera above the arenas and record for 5 min.</li>
	<li>Analyze the recorded movement trajectories using Ctrax software (freely available from Caltech)16. Export the tracked data in a programming language (e.g., Matlab)-compatible format and analyze the data based on distance traveled per frame17. Calculate the mean walking distance for each fly and combine it with all other recorded flies/group to obtain a mean cumulative distance travelled by the population of files in the same group.</li>
</ol>

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+neurobehavioral+sequelae+after+repetitive+mild+traumatic+brain+injury:+a+novel+mouse+model+of+chronic+traumatic+encephalopathy.">The spectrum of neurobehavioral sequelae after repetitive mild traumatic brain injury: a novel mouse model of chronic traumatic encephalopathy.</a> J Neurotrauma. 31 (13), 1211-1224 (2014).</li><li>Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster+in+the+study+of+human+neurodegeneration.">Drosophila melanogaster in the study of human neurodegeneration.</a> CNS Neurol Disord Drug Targets. 9 (4), 504-523 (2010).</li><li>Littleton, J. 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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Method+to+Inflict+Closed+Head+Traumatic+Brain+Injury+in+Drosophila.">A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila.</a> J Vis Exp. (e52905), (2015).</li><li>Branson, K., Robie, A. A., Bender, J., Perona, P., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+ethomics+in+large+groups+of+Drosophila.">High-throughput ethomics in large groups of Drosophila.</a> Nat Methods. 6 (6), 451-457 (2009).</li><li>Straw, A. D., Dickinson, M. 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This work was supported by the Johns Hopkins University School of Medicine faculty startup fund to L.C.

Animal models that faithfully model CTE features, including neurophysiological alterations, neuropathological hallmarks, and neurobehavioral deficits, are essential for uncovering disease mechanisms and for developing diagnostic and therapeutic targets. It is understandable that no animal model of a human disease is perfect at mimicking all clinically relevant endpoints. However, we believe that a robust CTE model should satisfy the following three requirements: (1) the impact must be directly applied to a head that has ...

Chronic Traumatic Encephalopathy (CTE) has recently been recognized as a distinct neurodegenerative disorder, separate from other tauopathies such as Alzheimer&#39;s disease1. Unlike Alzheimer&#39;s disease and other common tauopathies-whose most important risk factors are advancing age and a family history of dementia,&#160;CTE, as indicated by its name, implies a close association with a history of brain trauma, most likely seen in contact sports athletes, such as boxers and football players, as well as in military veterans2,3,4,5. It is thought to be initiated by repeated concussive and subconcussive blows to the head. Patients may present symptoms and signs such as cognitive deficits, mood and behavior changes, and movement dysfunction, which overlap significantly with Alzheimer&#39;s disease, frontotemporal dementia, Lewy body dementia, and Parkinson&#39;s disease6. In contrast, post-mortem examinations of brain tissue reveal a distinct pattern of hyperphosphorylated tau accumulation surrounding small blood vessels at the depths of the cortical sulci, a pathognomonic feature not observed in the other degenerative conditions7. However, so far, very little is known about the pathogenesis leading to disease manifestation. This is in large part due to the lack of a faithful animal model - only recently have rodent models been generated5,8. These model organisms have the disadvantages of cost-intensive care and a relatively long life span, which are not well-suited for neurodegenerative disease studies.
Compared to mammalian counterparts, invertebrate animals such as Drosophila&#160;are an excellent alternative, with their cost-effective maintenance, extensive tools for dissecting genetic determinants, and relatively short lifespan9. Remarkably, fly and human brains share evolutionarily conserved molecular and cellular pathways, as well as anatomical similarities10,11,12. Two ingenious Drosophila models to study traumatic brain injury have been reported previously13,14. The first &#34;High Impact Trauma&#34; (HIT) device designed by Katzenberger and colleagues contained free-moving flies in a plastic vial that was tied to the free end of a metal spring13,15. When the plastic vial was tilted upright and released, it hit a polyurethane pad and imparted trauma to the flies as they bounced to the vial wall and rebounded. In contrast, Barekat and colleagues designed a different delivery method using the Omni Bead Ruptor-24 homogenizer platform14. Flies were incapacitated with CO2 and placed in a 2&#160;mL screwcap tube that was secured to the homogenizer and subjected to preprogrammed shaking conditions. One benefit of using the tissue homogenizer system is that the experimenter could modulate the intensity of injury, duration of injury, and number of injury bouts. However, both regimes suffer the same drawback: primary injuries to the head are randomly inflicted in terms of impact location and strength. In addition, both methods resulted in considerable mortality, caused by inevitable collateral damage to other parts of the body and internal organs. Here, we describe a novel method to induce mTBI in fruit flies. Our apparatus consists of a gas-propelled ballistic impactor. Compared to the existing Drosophila models14,15, our method has the unique advantage of delivering measurable impact, directed only at the free-moving fly head, thus allowing for the accurate control of various factors, such as impact severity, the time interval between impacts, and the total number of impacts sustained.

<table><tbody><tr><td>Aerosol Barrier</td><td>USA Scientific</td><td>1120-8810</td><td>Used as an impactor</td></tr><tr><td>200 &amp;#956;L Pipette Tip</td><td>USA Scientific</td><td>1111-0706</td><td>Used as a fly head holder</td></tr><tr><td>1000 &amp;#956;L Pipette Tip</td><td>USA Scientific</td><td>1122-1830</td><td>Used as a connector</td></tr><tr><td>1 mL Tuberculin Syringe</td><td>Becton Dickinson</td><td>309625</td><td></td></tr><tr><td>60 mm Petri Dishes</td><td>Fisher Scientific</td><td>FB0875713A</td><td>Used as a tracking arenas</td></tr><tr><td>Flow Regulator</td><td>Genesee Scientific</td><td>59-122WC</td><td></td></tr><tr><td>Standard Clamp Holder/stand</td><td>EISCO Scientific</td><td>CH0688</td><td></td></tr><tr><td>Fine Brush</td><td>Genesee Scientific</td><td>59-204</td><td></td></tr><tr><td>Flypad</td><td>Genesee Scientific</td><td>59-114</td><td></td></tr><tr><td>Sylgard Silicone Elastomer</td><td>Dow Corning</td><td>4019862</td><td></td></tr><tr><td>CCD Camera</td><td>Microsoft&amp;nbsp;</td><td>HD-5000</td><td></td></tr><tr><td>Ctrax Walking Fly Tracker</td><td>Caltech</td><td>Ctrax 0.2.11</td><td></td></tr><tr><td>MATLAB Image Processing Toolbox</td><td>MATLAB</td><td>R2015b</td><td></td></tr></tbody></table>

a novel method to model chronic traumatic encephalopathy in drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

To establish a CTE model using adult Drosophila, we determined the effectiveness of our device at inflicting a single closed-head injury. To eliminate the variations relating to genotype, sex, or age, we used 2-day-old Canton-S WT female flies in the experiment. We could easily control the strength of the impactor by regulating the flow rate of CO2 at a constant gas pressure of 100 kPa. Flies exposed to a single strike at the highest flow rate (15 L/min) exhibited mini...

Watch this Scientific Journal Video about A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila at JoVE.com

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

a-novel-method-to-model-chronic-traumatic-encephalopathy-in-drosophila

Research

JoVE Journal

Neuroscience

8.2K Views.  Johns Hopkins University School of Medicine.  Hier beschrijven we een nieuwe aanpak om traumatisch hersenletsel in Drosophila melanogaster te veroorzaken . Onze methode heeft het voordeel om herhaalde effecten te leveren met instelbare sterkte aan het hoofd alleen. Verdere verkenning van het ongewervelde systeem zal helpen bij het verlichten van de pathogenese van chronische traumatische encefalopathie. 

Video: A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Developmental Biology

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the fly as an experimental system include its genetic tractability, short life span and the possibility to observe and quantitatively analyze complex behaviors. The expression of disease-linked genes in specific neuronal populations of the Drosophila brain, can be used to model human neurodegenerative diseases such as Parkinson's and Alzheimer's 5.
Dopaminergic (DA) neurons are among the most vulnerable neuronal populations in the aging human brain. In Parkinson's disease (PD), the most common neurodegenerative movement disorder, the accelerated loss of DA neurons leads to a progressive and irreversible decline in locomotor function. In addition to age and exposure to environmental toxins, loss of DA neurons is exacerbated by specific mutations in the coding or promoter regions of several genes. The identification of such PD-associated alleles provides the experimental basis for the use of Drosophila as a model to study neurodegeneration of DA neurons in vivo. For example, the expression of the PD-linked human &alpha;-synuclein gene in Drosophila DA neurons recapitulates some features of the human disease, e.g. progressive loss of DA neurons and declining locomotor function 2. Accordingly, this model has been successfully used to identify potential therapeutic targets in PD 8.
Here we describe two assays that have commonly been used to study age-dependent neurodegeneration of DA neurons in Drosophila: a climbing assay based on the startle-induced negative geotaxis response and tyrosine hydroxylase immunostaining of whole adult brain mounts to monitor the number of DA neurons at different ages. In both cases, in vivo expression of UAS transgenes specifically in DA neurons can be achieved by using a tyrosine hydroxylase (TH) promoter-Gal4 driver line 3, 10.

Assessing Neurodegenerative Phenotypes in Drosophila Dopaminergic Neurons by Climbing Assays and Whole Brain Immunostaining

Here we describe two assays that have been established to study age-dependent neurodegeneration of dopaminergic (DA) neurons in Drosophila: the climbing/startle-induced negative geotaxis assay which allows to study the functional effects of DA neurons degeneration and the tyrosine hydroxylase immunostaining which is used to identify and count DA neurons in whole brain mounts.

Drosophila melanogaster is a valuable model organism to study aging and pathological degenerative processes in the nervous system. The advantages of the ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Biology

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our understanding of the molecular mechanisms underlying axon degeneration remains limited. Injury-induced axon degeneration serves as a simple model to study how severed axons execute their own disassembly (axon death). Over recent years, an evolutionarily conserved axon death signaling cascade has been identified from flies to mammals, which is required for the separated axon to degenerate after injury. Conversely, attenuated axon death signaling results in morphological and functional preservation of severed axons and their synapses. Here, we present three simple and recently developed protocols that allow for the observation of axonal morphology, or axonal and synaptic function of severed axons that have been cut-off from the neuronal cell body, in the fruit fly Drosophila. Morphology can be observed in the wing, where a partial injury results in axon death side-by-side of uninjured control axons within the same nerve bundle. Alternatively, axonal morphology can also be observed in the brain, where the whole nerve bundle undergoes axon death triggered by antennal ablation. Functional preservation of severed axons and their synapses can be assessed by a simple optogenetic approach coupled with a post-synaptic grooming behavior. We present examples using a highwire loss-of-function mutation and by over-expressing dnmnat, both capable of delaying axon death for weeks to months. Importantly, these protocols can be used beyond injury; they facilitate the characterization of neuronal maintenance factors, axonal transport, and axonal mitochondria.

Morphological and Functional Evaluation of Axons and their Synapses during Axon Death in Drosophila melanogaster

Here, we provide protocols to perform three simple injury-induced axon degeneration (axon death) assays in Drosophila melanogaster to evaluate the morphological and functional preservation of severed axons and their synapses.

Axon degeneration is a shared feature in neurodegenerative disease and when nervous systems are challenged by mechanical or chemical forces. However, our ...

Tracking Freely Walking Flies: A Method to Assess Locomotion in Drosophila

Source: Sun, M. and Chen, L. L. <a href="https://www.jove.com/video/55602/" target="_blank">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila</a>. J. Vis. Exp. (2017).
This video describes how to track freely walking flies to assess their general locomotion ability. The featured protocol demonstrates the assay&#39;s setup and data analysis using video tracking software.

Source: Sun, M. and Chen, L. L. A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila. J. Vis. Exp. (2017).
This video describes how to ...

JoVE Encyclopedia of Experiments

Drosophila melanogaster (fruit fly)

Adult

A Technique to Inflict Closed-Head Traumatic Brain Injury in Drosophila Flies

Source: Sun, M., et al. <a href="https://app.jove.com/v/55602">A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila.</a> J. Vis. Exp. (2017).
This video demonstrates a procedure to inflict a closed-head traumatic injury in anesthetized Drosophila flies using a strike device with a gas-propelled impactor. The injury affects neuronal function and impairs walking ability.

1. Assembly of the Strike Device (Figure 1)

	Remove the plunger from a 1 mL tuberculin syringe. Cut the barrel at the 1 mL mark.
	Remove an aerosol ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Samenvatting

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