This work is supported by a Canadian Health Research Institute (CIHR) Team Grant to Dr. Bolduc (co-PI) and Dr. Guy Rouleau (PI). We would like to thank Dr. Oksana Suchowersky, Dr. Kathryn Todd, the members of the University of Alberta fly club and Dr. Clayton Dickson for help in development of the method and statistical analysis.

Research on Drosophila melanogaster was in compliance with the University of Alberta&#8217;s research guidelines.
1. Fly Collection
<ol>
	<li>Collect 20 flies using CO2 (g) anesthetization and place in a 25 mm x 95 mm collection vial containing food.</li>
	<li>Store vials containing flies horizontally to avoid trapping flies in any liquids that may accumulate in the bottom the vial.</li>
	<li>Incubate flies for at least 21 hr&#160;at 22 &#176;C at 45% humidity in an incubator for approximately for 15 hr. Set the incubator with a 12 hr&#160;light : dark cycle.</li>
</ol>
2. Climbing Assay
<ol>
	<li>The following morning, transfer 20 flies from a single vial into a 250 ml glass graduated cylinder. Mark the position of the cylinder to keep it constant everyday. Use one glass cylinder per genotype to prevent cross contamination between the genotypes. Wash at the end of each experiment and rotate them between genotypes.
	<ol>
		<li>Conduct the experiments in ambient light (or red light if there is a potential defect in vision) at temperature and humidity of 22 &#176;C and 40% respectively. To avoid circadian rhythm confound, always perform experiments at the same time of day.</li>
	</ol>
	</li>
	<li>Seal the top of cylinder with a barrier film (wax film) to prevent the escape of any flies (Figure 2).</li>
	<li>Set up the video camera on a tripod. Focus camera on the 190 ml line of the 250 ml graduated cylinder (17.5 cm).</li>
	<li>Count the number of dead flies at the bottom of the cylinder and in the food vials. Record this number as the mortality.</li>
	<li>Very lightly tap the cylinder against a closed cell foam pad repeatedly with enough force to displace the flies to the inner bottom surface. Tap 5 - 10 times while using the other hand to press record on the camera.</li>
	<li>Press the &#8220;Record&#8221; button on the camera.</li>
	<li>Start the video camera recording and tap the cylinder six times in a distinct non-rhythmic pattern.</li>
	<li>Conduct each trial for 2 min from the time the flies are last tapped and record the number of flies crossing the height of 17.5 cm (190 ml) at each time point chosen (quantify every 10 sec). Note: The ml marking on the cylinder will vary from one cylinder model to another depending on diameter. To avoid error, measure the height on each cylinder used.</li>
	<li>Once the trial has ended, dispose of flies in 95% ethanol.</li>
	<li>Repeat steps 2.1 to 2.9 until all the replicates have been tested with fresh flies every time. 
	Note: Although 5 replicates may be enough with a mutation having a strong effect on locomotion, 10 biological replicates of 20 flies (200 flies) is recommended to detect smaller differences.</li>
	<li>Upon completion of the experiment, wash the cylinders in the lab dishwasher and dry O/N to be re-used.</li>
</ol>
3. Analysis
<ol>
	<li>Analyze videos of each fly trial. Each 10 sec, record the total number of flies that pass the target line.
	<ol>
		<li>If a fly climbs back down or falls, record that fly as -1 and count the next fly to cross the target line as the same number as the fly that climbed back down or fell. For example, if the 15th fly falls below the target line, the next fly to cross the line (the 16th fly) is considered the 15th fly and not the 16th.</li>
	</ol>
	</li>
	<li>Subtract the mortality from the total number of flies (20) to obtain the number of flies that remain in the trial. At each time point, obtain the fraction of flies above the target line.</li>
	<li>Plot each percentage at each time point (see Figure 3).</li>
	<li>Analyze the performance at the 120 sec data point and perform student t-test when 2 groups are present or ANOVA and a post-test for multiple comparisons (with Bonferroni modification for planned and Tukey for unplanned comparisons). The Kolmogorov-Smirnov tests17 is also performed to ascertain normality and equal variance but also to compare the distributions of the mutant group to the control.</li>
	<li>To present the data over aging, plot the percentage of flies climbing at 120 sec with flies of different ages (2 days, 1 week, 2 weeks) to see if there is a progressive deficit (Figure 4).</li>
</ol>

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The authors have nothing to disclose.

Drosophila has already proven to be an excellent model in Parkinson&#8217;s disease14 and other neurodegenerative conditions1,2. In addition to the genetic tools available in Drosophila, its genome is highly conserved for genes involved in neurological disorders19. The advent of genome wide genetic screening methods (including whole exome sequencing) is likely to continue to provide a larger list of candidate genes associated with human movement disorders. The developmen...

Devastating neurodegenerative disorders such as Parkinson&#8217;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia are increasingly recognized. Unfortunately, most of these neurodegenerative disorders are still without treatments. The widespread clinical use of genome-wide, unbiased genetic tests such as whole exome sequencing has led to an increasing number of genes being implicated in human locomotive disorders. Despite this progress, the pathological progression from early to late stages, remains elusive in these disorders. Drosophila provides one with the genetic tools for studying gene requirement in a controlled spatial and temporal manner. In addition, Drosophila has proven useful in screening drugs for neurological conditions such as Parkinson&#8217;s1, Alzheimer&#8217;s2, intellectual disability3,4 and epilepsy5,6 among others. Our aim was to develop a cost effective and reliable assay that would allow high throughput analysis that would still be sensitive enough to detect small changes in motor performance.
There are several assays used to quantify the effects of genetic mutation and/or environmental condition on Drosophila climbing behavior. Most of the assays capitalize on the natural tendency of flies to climb, known as negative geotaxis, or the climbing assay. Benzer7 suggested in 1967 that the counter-current apparatus used for the study of phototaxis could also be used to study gravitaxis. Since then, Ganetsky8 and many others9-12 have built on the initial assay. The principle is to place a known number of flies in a vial and tap the vial strongly against a hard surface, causing the flies to fall to the bottom of the vial. As it is an innate behavior, the flies will attempt to climb to the top of the vial, opposed to gravity. This assay is quantitative and measures how many flies have climbed past a marker on the vial during an allotted time period. Measurement of speed instead of total number of flies climbing has become a reliable parameter and shown defects in cases where the number of flies criteria was not significant13.
The climbing assay has proven useful in the study of many neurodegenerative disorders including Parkinson&#8217;s disease14. However, we noted that locomotive defects may not be detectable at time where neurodegeneration is already seen in pathological studies14. Thus, use of the traditional assay may limit the ability to study the early stages of disease pathogenesis. The appearance of locomotive defects during later stages of pathology may reflect a disease whose progression is too advanced for complete rescue.
This raises a potential issue with the sensitivity of the traditional climbing assay. The potential inability of the traditional climbing assay to detect mild locomotive defects can be attributed to the height to which the flies are required to climb. The traditional assay15,16 measures the number of flies to successfully climb over a height of 2 to 5 cm in 10 to 20 sec.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Drosophila stocks</td>
			<td></td>
			<td></td>
			<td>The stocks are selected depending on the experiments. The temperature and humidity in the room and in the incubator must be controled and consistent to avoid flies being too staticky or too wet.</td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td></td>
			<td></td>
			<td>Any digital camcorder will do. Make sure they can focus on close object.</td>
		</tr>
		<tr>
			<td>Graduated cylinder</td>
			<td>Kimble</td>
			<td>20028W</td>
			<td>Different models of graduated cylinder may have different diameter. It is therefore imporant to measure the height.</td>
		</tr>
		<tr>
			<td>computer</td>
			<td></td>
			<td></td>
			<td>Any model will do. We used the computer to monitor the climbing of the flies and record the number of flies at each time point</td>
		</tr>
	</tbody>
</table>

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.

Climbing is a strong and reproducible behavior. Indeed, one day old wild-type flies reach the target distance climbing performance rapidly (25 - 30 sec). Mutant flies present a range of performance from mild (or delayed) to complete inability to climb to the target. We illustrate this here with two different mutant alleles. The first one is a severe allele of the gene spastin caused by a complete deletion of the spastin gene (spas5.75) 18. In this line (spas5.75 with TM6b) one day old fli...

Watch this Scientific Journal Video about Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders at JoVE.com

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

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 ...

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JoVE Journal

Neuroscience

18.3K Views.  University of Alberta. 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. 

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

Ex vivo Culturing of Whole, Developing Drosophila Brains

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

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

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 ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...