We thank members of the Kopito, Luo, and Pearce labs for many helpful discussions during development of these methods. We also thank Brian Temsamrit for critical reading of this manuscript. This work was supported by funds from University of the Sciences and the W.W. Smith Charitable Trusts.

The authors have nothing to disclose.

As the numbers of patients suffering from neurodegenerative diseases continues to increase, there is an urgent need to increase the understanding of the molecular pathogenesis of these diseases so that better therapies can be developed. Here, we describe methods that allow for monitoring prion-like transmission of pathogenic protein aggregates between different cell types in the model organism, Drosophila melanogaster. We have recently used this methodology to demonstrate prion-like transmission of mutant Htt ag...

The prion hypothesis states that the infectious agent responsible for the transmissible spongiform encephalopathies (e.g., Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in deer and elk, and &#34;mad cow disease&#34; in cattle) is solely composed of protein and devoid of nucleic acids1. In prion diseases, the cellular prion protein (PrPC) assumes a non-native, stable fold (PrPSc) that is highly beta sheet-rich and can self-propagate by converting and recruiting monomeric PrPC molecules into stable amyloid aggregates. PrPSc aggregates use this self-replicating mechanism to spread between different cells in an organism and even between individual organisms2.
Protein misfolding and aggregation is also a central feature of most neurodegenerative diseases (Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS))3. Formation of intra- or extra-cellular aggregated protein assemblies in these diseases is closely associated with cytotoxicity4 and progresses along highly reproducible and disease-specific paths through the brain over time5,6. These patterns of spread suggest that pathogenic aggregates associated with these disorders have prion-like properties. Strong support now exists for prion-like transmission of aggregates associated with AD, PD, HD, and ALS - they spread from cell-to-cell and template the conformational change of monomeric forms of the same protein in previously unaffected cells7,8.
The majority of studies investigating prion-like spread of protein aggregates to date have been performed using mammalian cell culture models, where aggregates transfer into the cytoplasm of na&#239;ve cells from the extracellular space or from another cell&#39;s cytoplasm9,10,11,12,13,14,15, or by injecting aggregate-containing material into mouse brains and monitoring aggregate appearance outside of the injection site16,17,18,19,20,21,22,23. More recently, transgenic animals have been used to demonstrate that intracellular aggregates spread to other cells within intact brains24,25,26,27,28,29,30. Here, we describe a method for direct visualization of aggregate transfer between individual cells in the intact brain of Drosophila melanogaster. Drosophila models of HD/polyglutamine (polyQ) diseases were first developed nearly two decades ago31,32 and have provided many invaluable insights into the pathogenic mechanisms that underlie these disorders33. HD is an inherited neurodegenerative disorder caused by an autosomal dominant mutation in the gene that codes for the protein huntingtin (Htt)34. This mutation results in expansion of a polyQ stretch near Htt&#39;s N-terminus beyond a pathogenic threshold of ~ 37 glutamines, causing the protein to misfold and aggregate35,36. Wild-type Htt proteins containing &#60;37 glutamines in this stretch achieve their native fold, but can be induced to aggregate upon direct physical contact with a Htt aggregate &#34;seed&#34;12,27,37. We exploit this homotypic, nucleated aggregation of wild-type Htt as a readout for prion-like transfer and cytoplasmic entry of mutant Htt aggregates originating in other cells.
Determining the mechanisms by which prion-like aggregates travel between cells can lead to the identification of novel therapeutic targets for incurable neurodegenerative diseases. We take advantage of the rapid life cycle, ease of use, and genetic tractability of Drosophila melanogaster to define molecular mechanisms for cell-to-cell spread of mutant Htt aggregates. Our experimental strategy employs two binary expression systems available in Drosophila, the well-established Gal4-specific upstream activating sequence (Gal4-UAS) system38 and the recently-developed QF-QUAS system39. Coupling these two independent systems allows restricting expression of mutant and wild-type Htt transgenes to distinct cell populations within the same fly40. Using this approach, we examine prion-like spreading of mutant Htt by monitoring the redistribution of cytoplasmic wild-type Htt from its normally diffuse, soluble state to an aggregated state, a direct consequence of physical contact with a pre-formed mutant Htt aggregate &#34;seed.&#34; Conversion of wild-type Htt by mutant Htt can be confirmed using biochemical or biophysical techniques that report protein-protein interactions, such as fluorescence resonance energy transfer (FRET)9,27,41.
Importantly, we can also access a large number of genetic tools in Drosophila to identify genes and/or pathways that mediate prion-like spread of protein aggregates. We have recently used this approach to unveil a key role for the cell surface scavenger receptor, Draper42,43, in transferring mutant Htt aggregates from neuronal axons to nearby phagocytic glia in the Drosophila central nervous system (CNS)27. Thus, the genetic- and imaging-based approach that we describe here can reveal important basic biological information about a disease-relevant phenomenon in the simple-to-use but powerful model organism, Drosophila.

<table><tbody><tr><td>Phosphate buffered saline (PBS), 10X, pH 7.4</td><td>ThermoFisher Scientific</td><td>AM9625</td><td>Dilute to 1X</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284-1L</td><td></td></tr><tr><td>Kimwipes</td><td>Thomas Scientific</td><td>2904F24</td><td></td></tr><tr><td>20% paraformaldehyde (PFA)</td><td>Electron Microscopy Sciences</td><td>15713-S</td><td></td></tr><tr><td>Normal Goat Serum (NGS), filtered</td><td>Lampire Biological Laboratories</td><td>7332500</td><td>Aliquot and freeze upon receipt</td></tr><tr><td>Chicken anti-GFP</td><td>Aves Labs</td><td>GFP-1020</td><td>Use at 1:500 dilution</td></tr><tr><td>Rabbit anti-DsRed</td><td>Clontech</td><td>632496</td><td>Use at 1:2000 dilution; can recognize DsRed-based fluorescent proteins (e.g. mCherry, mStrawberry, tdTomato, etc.)</td></tr><tr><td>Mouse anti-Bruchpilot</td><td>Developmental Studies Hybridoma Bank</td><td>nc82</td><td>Use at 1:100 dilution; will label active pre-synaptic structures thoughout the fly brain</td></tr><tr><td>FITC anti-chicken</td><td>ThermoFisher Scientific</td><td>SA1-7200</td><td>Use at 1:250 dilution</td></tr><tr><td>Alexa Fluor 568 anti-rabbit</td><td>Life Technologies</td><td>A11011</td><td>Use at 1:250 dilution</td></tr><tr><td>Alexa Fluor 647 anti-mouse antibody</td><td>Life Technologies</td><td>A21235</td><td>Use at 1:250 dilution</td></tr><tr><td>Slowfade Gold Antifade Reagent</td><td>Life Technologies</td><td>S36936</td><td></td></tr><tr><td>Microscope Slides (25 x 75 x 1.0 mm)</td><td>Fisher Scientific</td><td>12-550-143</td><td></td></tr><tr><td>Cover Glass (22 x 22 mm)</td><td>Globe Scientific</td><td>1404-15</td><td></td></tr><tr><td>Dumont Biology Grade Forceps, Style 3</td><td>Ted Pella</td><td>503</td><td>use in non-dominant hand</td></tr><tr><td>Dumont Biology Grade Forceps, Style 5</td><td>Ted Pella</td><td>505</td><td>use in dominant hand</td></tr><tr><td>LAS X image analysis software</td><td>Leica</td><td></td><td></td></tr><tr><td>Imaris image analysis software</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

monitoring cell-to-cell transmission of prion-like protein aggregates in drosophila melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), Huntington&#39;s disease (HD), and amyotrophic lateral sclerosis (ALS). Protein aggregates are closely associated with neuropathology in these diseases, although the exact mechanism by which aberrant protein aggregation disrupts normal cellular homeostasis is not known. Emerging data provide strong support for the hypothesis that pathogenic aggregates in AD, PD, HD, and ALS have many similarities to prions, which are protein-only infectious agents responsible for the transmissible spongiform encephalopathies. Prions self-replicate by templating the conversion of natively-folded versions of the same protein, causing spread of the aggregation phenotype. How prions and prion-like proteins in AD, PD, HD, and ALS move from one cell to another is currently an area of intense investigation. Here, a Drosophila melanogaster model that permits monitoring of prion-like, cell-to-cell transmission of mutant huntingtin (Htt) aggregates associated with HD is described. This model takes advantage of powerful tools for manipulating transgene expression in many different Drosophila tissues and utilizes a fluorescently-tagged cytoplasmic protein to directly report prion-like transfer of mutant Htt aggregates. Importantly, the approach we describe here can be used to identify novel genes and pathways that mediate spreading of protein aggregates between diverse cell types in vivo. Information gained from these studies will expand the limited understanding of the pathogenic mechanisms that underlie neurodegenerative diseases and reveal new opportunities for therapeutic intervention.

The methods described here produce robust data demonstrating prion-like transfer of Htt protein aggregates from one cell population to another in the intact fly CNS. Conversion of wild-type Htt from diffuse to punctate is observed by direct fluorescence of this YFP fusion protein in recipient glia as a result of HttQ91-mCherry expression in donor ORNs (Figure 2A-C and Figure 4A, B). Accurate repo...

Watch this Scientific Journal Video about Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster at JoVE.com

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

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Neuroscience

7.9K Views.  University of the Sciences. Accumulating evidence supports the idea that pathogenic protein aggregates associated with neurodegenerative diseases spread between cells with prion-like properties. Here, we describe a method that enables visualization of cell-to-cell spreading of prion-like aggregates in the model organism, Drosophila melanogaster.

Video: Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such as Chronic Wasting Disease (CWD) of cervids and scrapie in sheep. The Prion hypothesis asserts that this abnormal conformer constitutes most or all of the infectious prion. The role of the immune system in early events in peripheral prion pathogenesis has been convincingly demonstrated for CWD and scrapie 1-3. Transgenic and pharmacologic studies in mice revealed an important role of the Complement system in retaining and replicating prions early after infection 4-6. In vitro and in vivo studies have also observed prion retention by dendritic cells 7-10, although their role in trafficking remains unclear 11-16. Macrophages have similarly been implicated in early prion pathogenesis, but these studies have focused on events occurring weeks after infection 3,11,17. These prior studies also suffer from the problem of differentiating between endogenous PrPC and infectious prions. Here we describe a semiquantitative, unbiased approach for assessing prion uptake and trafficking from the inoculation site by immune cells recruited there. Aggregated prion rods were purified from infected brain homogenate by detergent solubilization of non-aggregated proteins and ultracentrifugation through a sucrose cushion. Polyacrylamide gel electrophoresis, coomassie blue staining and western blotting confirmed recovery of highly enriched prion rods in the pelleted fraction. Prion rods were fluorochrome-labeled then injected intraperitoneally into mice. Two hours later immune cells from peritoneal lavage fluid, spleen and mediastinal and mesenteric lymph nodes were assayed for prion rod retention and cell subsets identified by multicolor flow cytometry using markers for monocytes, neutrophils, dendritic cells, macrophages and B and T cells. This assay allows for the first time direct monitoring of immune cells acquiring and trafficking prions in vivo within hours after infection. This assay also clearly differentiates infectious, aggregated prions from PrPC normally expressed on host cells, which can be difficult and lead to data interpretation problems in other assay systems. This protocol can be adapted to other inoculation routes (oral, intravenous, intranervous and subcutaneous, e.g.) and antigens (conjugated beads, bacterial, viral and parasitic pathogens and proteins, egg) as well.

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Presence of an abnormal form a host-encoded prion protein (PrPC) that is protease resistant, pathologic and infectious characterizes prion diseases such ...

Immunology and Infection

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 Technique to Assay Locomotor Activity in Drosophila

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and diverse behavioral features. Identifying behavioral deficiency in animal models is a crucial measure of disease severity, for example, in neurodegenerative diseases where patients often experience impairments in motor function. However, with the availability of various systems to track and assess motor deficits in fly models, such as drug-treated or transgenic individuals, an economical and user-friendly system for precise evaluation from multiple angles is still lacking. A method based on the AnimalTracker application programming interface (API) is developed here, which is compatible with the Fiji image processing program, to systematically evaluate the movement activities of both adult and larval individuals from recorded video, thus allowing for the analysis of their tracking behavior. This method requires only a high-definition camera and a computer peripheral hardware integration to record and analyze behavior, making it an affordable and effective approach for screening fly models with transgenic or environmental behavioral deficiencies. Examples of behavioral tests using pharmacologically treated flies are given to show how the techniques can detect behavioral changes in both adult flies and larvae in a highly repeatable manner.

A Simple Technique to Assay Locomotor Activity in Drosophila

The present protocol assesses the locomotor activity of Drosophila by tracking and analyzing the movement of flies in a hand-made arena using open-source software Fiji, compatible with plugins to segment pixels of each frame based on high-definition video recording to calculate parameters of speed, distance, etc.

Drosophila melanogaster is an ideal model organism for studying various diseases due to its abundance of advanced genetic manipulation techniques and ...

Behavior

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 ...

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 ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Biology

Monitoring Epileptiform Events in Drosophila via Calcium Imaging

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals worldwide. Despite the clinical importance of epilepsy, the functional analysis of neural activity in the central nervous system is still to be developed. Recent advancements in imaging technology, in combination with stable expression of genetically encoded calcium indicators, such as GCaMP6, have revolutionized the study of epilepsy at both brain-wide and single-cell resolution levels. Drosophila melanogaster has emerged as a tool for investigating the molecular and cellular mechanisms underlying epilepsy due to its sophisticated molecular genetics and behavioral assays. In this study, we present a novel and efficient protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The whole brain is prepared from cac, a well-known epilepsy gene, knockdown flies for calcium imaging with a confocal microscope to identify the neural activity as a follow-up to the bang-sensitive seizure-like behavior assay. The cac knockdown flies showed a higher rate of seizure-like behavior and abnormal calcium activities, including more large spikes and fewer small spikes than wild-type flies. The calcium activities were correlated to seizure-like behavior. This methodology serves as an efficient methodology in screening the pathogenic genes for epilepsy and exploring the potential mechanism of epilepsy at the cellular level.

Author Spotlight: Insights into the Techniques and Findings of Recent Advancements in Epilepsy Research 

Here, we present a protocol for ex vivo calcium imaging in GCaMP6-expressing adult Drosophila to monitor epileptiform activities. The protocol provides a valuable tool for investigating ictal events in adult Drosophila through ex vivo calcium imaging, allowing for exploration of the potential mechanisms of epilepsy at the cellular levels.

Epilepsy is a neurological disorder characterized by recurrent seizures, partially correlated with genetic origin, affecting over 70 million individuals ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

Time-Lapse Imaging to Monitor the Transcellular Spreading of Prion-Like Proteins in Transgenic Nematodes

Source: Nussbaum-Krammer, C. I., et al. <a href="https://app.jove.com/v/52321">Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans.</a> J. Vis. Exp. (2015)
This video demonstrates the procedure for in vivo imaging of C. elegans expressing prion-like proteins tagged with red fluorescent protein. The protocol involves immobilizing nematodes on an agarose pad for time-lapse confocal microscopy, revealing protein aggregation and transport within and between cells. This technique is crucial for studying protein aggregation related to neurodegenerative diseases.

Obrazowanie poklatkowe w celu monitorowania transkomórkowego rozprzestrzeniania się białek podobnych do prionów u nicień transgenicznych

1. Monitoring Transcellular Spreading of Prion-like Proteins By In Vivo Time-lapse Imaging
NOTE: Grow C. elegans wild-type (WT) (N2) and transgenic lines ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

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Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

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

A Simple Technique to Assay Locomotor Activity in Drosophila

Mass Histology to Quantify Neurodegeneration in Drosophila

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

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Ex Vivo Calcium Imaging for Drosophila Model of Epilepsy

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Obrazowanie poklatkowe w celu monitorowania transkomórkowego rozprzestrzeniania się białek podobnych do prionów u nicień transgenicznych