This work was supported by grants to D.K. from the Medical Research Foundation of Oregon and from NIH/NINDS (NS047663). E.S. was supported by a training grant from the NIH (T32AG023477).

1. Fixing the Head on Collars and Embedding in Paraffin
Note: All of the steps in the fixation process should be done in a fume hood. Methylbenzoate, while not posing a health risk, has a highly distinct odor, which can be overwhelming if not handled in a fume hood.
<ol>
	<li>Before anesthetizing the flies, make up 50 mL of Carnoy solution by adding 15 mL of chloroform and 5 mL of glacial acetic acid to 30 mL of 99% ethanol (do not mix the chloroform and acetic acid). Pour it in a glass container with a flat bottom, such as a crystalizing dish, to ensure that the collars can lay flat and are completely covered by the solution.</li>
	<li>Anesthetize the flies with CO2 or ether.</li>
	<li>Thread flies (up to 20 with most collars) by their necks into the collars using forceps. Remember to align all of the heads in the same orientation, as seen in Figure 1A, and be gentle to ensure that no damage occurs to the head or eyes.</li>
	<li>Include sine oculis flies (arrows, Figure 1A) at random positions so that the order of the flies can be easily identified in the sections. In addition, if the experimental flies have a light or white eye color, thread some red-eyed flies, such as wild type, between them to ensure that sufficient pigment is present to stain the slide. Record the order of the flies on a protocol sheet together with the collar number if using more than one collar.</li>
	<li>Once a collar has been finished, place it in the prepared Carnoy solution for 3.5 - 4 h.</li>
	<li>Dump out the Carnoy solution into the appropriate disposal canister and begin the ethanol washes. Make sure to pour slowly so as not to disturb the placement of the collars in the container.</li>
	<li>Wash the collars for 30 min in 99% ethanol twice.</li>
	<li>Wash the collars in 100% ethanol for 1 h. Be sure to change the washes on time to prevent overdehydration.</li>
	<li>Put the collars in methylbenzoate O/N at RT. Seal the container with parafilm to prevent the evaporation of the methylbenzoate.</li>
	<li>Pour the methylbenozate into the proper disposable container in the fume hood. Add a previously-prepared mixture of 1:1 low melting point (56 - 57 &#176;C) paraffin wax and methylbenzoate for 1h. From this point on, the collars need to be kept in an incubator at 65 &#176;C to make sure that the paraffin does not harden.</li>
	<li>Pour out the methylbenozate and paraffin mixture into the proper disposable container, and pour molten pure paraffin wax, kept at 65 &#176;C, onto the collars.</li>
	<li>Change the paraffin after 30 min and repeat this at least 5 times. At least 6 - 8 washes should be performed.</li>
	<li>Once the washes are complete, place the collars into a rubber ice cube tray with slots approximately the size of the collars. Pour molten paraffin over them until completely covered and allow it to harden O/N (try to avoid air bubbles).</li>
	<li>Remove the paraffin blocks containing the collars from the ice cube tray. Separate the paraffin block from the collar using a razor blade, gently breaking off the collar. The heads will be in the paraffin block while the bodies will stay in the collar. The blocks can be kept at room temperature.</li>
	<li>To clean the collars, soak them in a deparafinization agent at 65 &#176;C to remove the paraffin, clean with light scrubbing, and wash in ethanol before reusing.</li>
</ol>
2. Sectioning and Mounting
<ol>
	<li>Warm a heating plate to 50 &#176;C. Place the object holders (or metal mounting blocks) and razor blades on the plate and let them warm up.</li>
	<li>Depending on the desired orientation for sectioning, attach the paraffin block either with the heads towards the side (for horizontal sections) or facing upwards (for frontal sections) to a heated mounting block (briefly melting the block at the contact side). Remove the block from the heating plate and allow it to cool for at least 10 min to ensure that the paraffin is hardened enough for a proper seal onto the mounting block. Keep the row of heads aligned in parallel with the surface of the block as much as possible to prevent uneven sections.</li>
	<li>Take a razor blade and trim the excess paraffin away from the fly heads so that only a small row with the embedded heads remains (the razor blade can be warmed up for easier trimming). Make sure not to trim too much so that the paraffin does not break during sectioning (more trimming can be done during sectioning).</li>
	<li>Place the mounting block into the object holder of the microtome and ensure that the alignment of the row of heads is as parallel as possible to the edge of the blade.</li>
	<li>Prepare microscope slides by covering them with a thin layer of poly-L-Lysine (PLL) solution and let them dry for 5 min. Cover them with water shortly before use.</li>
	<li>Cut 7&#160;&#181;m sections and transfer the ribbon of sections to the slide floating on the water. 
	NOTE: To obtain the entire brain for horizontal sections, we collect the ribbon from when starting to cut into the eye until the head has been completely cut (cutting from the proboscis into the brain). More than one slide may be needed for the entire head.</li>
	<li>Place the slide on a heat plate at 37 &#176;C and allow the ribbon to expand for about 1 min.</li>
	<li>Remove excess water (by pouring it off or using a tissue) and dry the slides O/N.</li>
	<li>Remove the paraffin wax from the slide by placing the slides in a tall, vertical slide-staining jar filled with a deparafinization agent (completely covering the sections). Perform 3 washes of 30 min - 60 min each.</li>
	<li>Remove the slide from the final wash. Place 2 drops of embedding media onto the slide and cover it with a large coverslip.</li>
</ol>
3. Photographing and Analyzing the Sections
<ol>
	<li>Allow the prepared slides to dry for 1 - 2 d. Then, examine them on a fluorescence microscope under blue light.</li>
	<li>Use a lower magnification to determine the orientation of the flies and to find the region of interest if focusing on a specific region. 
	NOTE: For the sws flies (see Figure 2), we find the section that contains the great commissure and take an image (usually at 40X magnification). When analyzing the entire brain (as in Figure 3), we scroll through all the sections from a head and either photograph the section with the most severe phenotype or all sections that show vacuoles.</li>
	<li>For a double-blind analysis, take and number images without knowing the genotype and record the collar number and position of the head in the row to identify them later.</li>
	<li>Once the images have been taken, analyze them using an imaging software.</li>
	<li>Count the number of vacuoles per section or per head. To measure the vacuole size, open the images in a software program and select the vacuoles with a selection tool. Determine the amount of pixels in the selected vacuoles.</li>
	<li>For conversion to &#181;m2, take a photo of a stage micrometer calibration slide at the magnification used for acquiring photos. Determine the amount of pixels in 100 &#181;m2 to calculate a conversion factor.</li>
	<li>Convert the total pixel number into &#181;m2 by dividing the number of pixels by the conversion factor calculated in the above step.</li>
</ol>

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The authors have nothing to disclose.

The described method provides a means to quantify neurodegeneration in the brain of Drosophila. While other methods, like counting a specific cell type, can be used to identify neurodegeneration, the advantage of this method is that it can be applied more generally. Counting cells requires that these cells can be reliably identified using either a specific antibody or the expression of a cell-specific marker, which is not always available. Furthermore, it has been shown that dramatically different results can be...

With the increase in life expectancy, neurodegenerative diseases like Alzheimer&#39;s or Parkinson&#39;s have become an increasing health threat for the general population. According to the National Institutes of Health, 115 million people worldwide are predicted to be affected by dementia in 2050. Although significant progress has been made in identifying genes and risk factors involved in at least some of these diseases, for many of them, the underlying molecular mechanisms are still unknown or not well understood.
Simple invertebrate model organisms like Caenorhabditis elegans and Drosophila melanogaster offer a variety of experimental advantages to study the mechanisms of neurodegenerative diseases, including a short life cycle, large number of progeny, and the availability of well-established and sometimes unique genetic and molecular methods1-12. Furthermore, these organisms are amenable to unbiased interaction screens that can identify factors contributing to these diseases by their aggravating or ameliorating effects on neurodegenerative phenotypes.
Analyzing such genetic interactions and assessing aging effects requires quantitative protocols to detect neurodegeneration and to measure its severity. This assessment can be done relatively easily when measuring behavioral aspects in Drosophila, such as olfactory learning, negative geotaxis, or fast phototaxis, which provide a numeric performance value13-21. It is also possible to determine the effects on neuronal survival by counting neurons. However, this is only possible when focusing on a specific population that is clearly identifiable, like the dopaminergic neurons that are affected in PD, and even then, the results have been controversial22-24.
The protocol described here uses the collar method to perform paraffin serial sections, a method that was originally developed by Heisenberg and B&#246;hl, who used it to isolate anatomical brain mutants in Drosophila25. The use of the collar method has subsequently been adapted, including in cryosections, vibratome sections, and plastic sections26-28. Here, this method is employed to obtain serial sections of the entire fly head, which can then be used to measure the vacuoles that develop in flies with neurodegenerative phenotypes16,21,29-32. These measurements can be done in specific brain areas or can cover the entire brain; the latter approach allows one to identify even weak degenerative phenotypes, as observed during aging. Finally, when using the collars, up to 20 flies can be processed as one preparation, which is not only less time-consuming, but also allows for the analysis of control and experimental flies in the same preparation, minimizing artifacts due to slight changes in the preparation.

<table><tbody><tr><td>Name of the Reagent/Equipment</td><td>Company</td><td>Catalog Number</td><td></td></tr><tr><td>Collar</td><td>Genesee Scientific</td><td>TS 48-100</td><td>We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A description on how to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html&amp;nbsp;</td></tr><tr><td>Rubber ice cube tray for embedding</td><td>Household store</td><td></td><td>The size can be made-to-fit by glueing in additional walls&amp;nbsp;</td></tr><tr><td>Crystallizing dish</td><td>Fisher Scientific company</td><td>08-762-3</td><td></td></tr><tr><td>Ether</td><td>Fisher Scientific Company</td><td>E138-1</td><td></td></tr><tr><td>Ethanol</td><td>Decon Laboratories Inc.</td><td>2701</td><td></td></tr><tr><td>Choloroform</td><td>Fisher Scientific Company</td><td>C298-500</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Fisher Scientific Company</td><td>A38-212</td><td></td></tr><tr><td>Methylbenzoate</td><td>Fisher Scientific Company</td><td>M205-500</td><td>Distinct Odor - Use in fume hood!</td></tr><tr><td>Low Melting Point Paraffin Wax</td><td>Fisher Scientific Company</td><td>T565</td><td>Make sure to keep extra melted in a 65&amp;deg;C waterbath</td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td>Reichert Jung 2040 Autocut</td><td></td></tr><tr><td>Microscope Slide</td><td>Fisher Scientific Company</td><td>12-550D</td><td></td></tr><tr><td>Microscope Cover Glass</td><td>Fisher Scientific Company</td><td>12-545-M</td><td></td></tr><tr><td>SafeClear</td><td>Fisher Scientific Company</td><td>314-629</td><td>Three different containers for washes</td></tr><tr><td>Vertical Staining Jar with Cover</td><td>Ted Pella Inc.&amp;nbsp;</td><td>432-1</td><td></td></tr><tr><td>Permount</td><td>Fisher Scientific Company</td><td>SP15-500</td><td></td></tr><tr><td>Poly-L-Lysine Solution (PLL)</td><td>Sigma Life Science</td><td>P8290-500</td><td></td></tr></tbody></table>

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.

Using the described method results in serial sections stained by the eye pigment33 that encompass the entire fly head. A part of this is shown in Figure 1B, where the sections from an individual head are shown from top to bottom. The sections from different flies are seen left to right in this example. To facilitate orientation and identification of the flies, an eyeless fly (sine oculis) is inserted as a marker at position 3 (arrow, Figure 1B<...

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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.

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 ...

mass-histology-to-quantify-neurodegeneration-in-drosophila

Research

JoVE Journal

Neuroscience

10.1K Views.  Oregon Health & Sciences University.  Drosophila wordt veel gebruikt als een modelsysteem om neurodegeneratie. Dit protocol beschrijft een methode waarbij degeneratie, zoals bepaald door vacuole-vorming in de hersenen, kan worden gekwantificeerd. Het minimaliseert ook effecten als gevolg van de experimentele procedure van de verwerking en snijden controle en experimentele vliegen als een monster. 

Video: Mass Histology to Quantify Neurodegeneration 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 ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Genetics

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 ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

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 ...

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused on neuromuscular junctions (NMJs) in Drosophila larvae, assessing synaptic integrity in adult Drosophila has received much less attention. Here we provide a straightforward method for dissection of the dorsal longitudinal muscles (DLMs), which are required for flight ability. In addition to flight as a behavioral readout, this dissection allows for the both DLM synapses and muscle tissue to be amenable to structural analysis using fluorescently labeled antibodies for synaptic markers or proteins of interest. This protocol allows for the evaluation of the structural integrity of synapses in adult Drosophila during aging to model the progressive, age-dependent nature of most neurodegenerative diseases.

Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration

The goal of this procedure is to dissect the dorsal longitudinal muscle (DLM) tissue to assess the structural integrity of DLM neuromuscular junctions (NMJs) in neurodegenerative disease models using Drosophila melanogaster.

Drosophila serves as a useful model for assessing synaptic structure and function associated with neurodegenerative diseases. While much work has focused ...

Direct Cryosectioning of Drosophila Heads for Enhanced Brain Fluorescence Staining and Immunostaining

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression patterns. Traditional methods often involve challenges such as performing complex dissection, maintaining tissue integrity, and visualizing specific expression patterns during high-resolution imaging. We present an optimized protocol that combines cryosectioning with fluorescence staining and immunostaining. This method improves tissue preservation and signal clarity and reduces the need for laborious dissection for Drosophila brain imaging. The method entails rapid dissection, optimal fixation, cryoprotection, and cryosectioning, followed by fluorescent staining and immunostaining. The protocol significantly reduces tissue damage, enhances antibody penetration, and yields sharp, well-defined images. We demonstrate the effectiveness of this approach by visualizing specific neural populations and synaptic proteins with high fidelity. This versatile method allows for the analysis of various protein markers in the adult brain across multiple z-planes and can be adapted for other tissues and model organisms. The protocol provides a reliable and efficient tool for researchers conducting high-quality immunohistochemistry in Drosophila neurobiology studies. This method's detailed visualization facilitates comprehensive analysis of neuroanatomy, pathology, and protein localization, making it particularly valuable for neuroscience research.

This study presents a simplified protocol for tissue processing involving decapitation, fixation, cryosectioning, fluorescence staining, immunostaining, and imaging, which can be extended to confocal and multiphoton imaging. The method maintains efficacy comparable to complex dissections, bypassing the need for advanced motor skills. Quantitative image analysis provides extensive investigative potential.

Immunostaining Drosophila melanogaster brains is essential for exploring the mechanisms behind complex behaviors, neural circuits, and protein expression ...

Assessing the Structural Integrity of the Dorsal Longitudinal Muscle Neuromuscular Junctions in Drosophila

Source: Sidisky, J. M., et al. <a href="https://app.jove.com/v/61363">Visualizing Synaptic Degeneration in Adult Drosophila in Association with Neurodegeneration.</a> J. Vis. Exp. (2020).
This video demonstrates a protocol for assessing the structural integrity of neuromuscular junctions in the dorsal longitudinal muscles of Drosophila. The procedure involves staining neurons and muscles with two distinct antibodies and comparing fluorescence patterns between mutant and wild-type flies. A reduction in neuronal staining in mutant flies indicates neurodegeneration, while the muscle structure remains intact.

1. Flash freezing and thorax bisection

	Before beginning the bisections, fill a Dewar flask with liquid nitrogen wearing proper cryo-protective gloves ...

Mass Histology to Quantify Neurodegeneration in Drosophila

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