This work was supported by a grant funded by the Korean government (MSIP) (NRF-2014K1A4A7A01074642), and the National Research Foundation of Korea (NRF) individual scientist support program (NRF-2013M3A9B5076486/NRF-2015R1D1A1A09057239).

1. Lentivirus production
NOTE: The production and manipulation of lentiviral vectors was carried out according to the National Institutes of Health (NIH) guidelines for research involving recombinant DNA. The plasmid encoding the wild-type and A4V mutant SOD1 tagged with enhanced YFP (SOD1WT-YFP and SOD1A4V-YFP) are described in Kim et al.11 Both gene fusion products were amplified using the PCR primer pair 5&#8242;-ATCGTCTAGACACCATGGCGACGAAGGTCGTGTGC-3&#8242; and 5&#8242;-TAGCGG CCGCTACTTGTACAGCTCGTCCATGCC-3&#8242;and inserted into the pTRIP-delta U3 CMV plasmid19 using XhoI and BsrGI restriction sites. In prior experiments, the HEK-293T cells were split at a ratio of 1:4 to 1:6 every two days. Avoidance of more than 20 passages and maintenance of cells at less 60% confluency helps to ensure good transfection efficiencies.
<ol>
	<li>On Day 1, split HEK-293T cells at 30 - 40% confluence in a 10-cm diameter tissue culture plate (3 x 106 cells/dish) in 10 mL of cell culture medium (high-glucose DMEM with 10% FBS and 4 mM L-glutamine) for virus production.</li>
	<li>Keep the 10-cm diameter tissue culture plate in a CO2 incubator (37 &#176;C, 5% CO2) overnight.</li>
	<li>On Day 2, prepare a DNA transfection solution containing 10 &#181;g of lentiviral vectors (pTRIP-delta U3 CMV - SOD1WT-YFP or -SOD1A4V-YFP) together with the lentiviral packaging plasmids (5 &#181;g of pVSVg; 10 &#181;g of pCMV-dR8.71) (Figure 2A)20. Add 125 &#181;L of 1 M CaCl2 and adjust the volume to 500 &#181;L using distilled water. Gently add 500 &#181;L of 0.05 M HEPES into the mixture and incubate for 10 min at room temperature.</li>
	<li>Replace HEK-293T cells medium with 10 mL pre-warmed fresh culture medium antibiotic-free mixed with 1 mL DNA transfection solution and incubate overnight at 37 &#176;C, 5% CO2.</li>
	<li>On Day 3, replace the cell supernatant with fresh culture medium.</li>
	<li>On Day 4, harvest the supernatant in a 15 mL tube and centrifuge for 5 min at 500 x g to remove the dead cells and debris. Further purify the supernatant by passing it through a 0.45 &#181;m filter using a large 60 mL syringe. Immediately dispense into single-use aliquots (300 &#181;L), and store at -80 &#176;C. To maintain maximum product activity, avoid a freeze-thaw cycle.</li>
</ol>
2. Lentiviral transduction
<ol>
	<li>Split the cell line to be infected at 60% confluence (HEK-293 cells and SH-SY5Y; 5 x 105 cells per well and U2OS; 1 x 105 cells per well) in a 6-well tissue culture plate in 2 mL of DMEM media supplemented with 10% FBS.</li>
	<li>Keep the 6-well tissue culture plate in a 37 &#176;C incubator at 5% CO2 overnight.</li>
	<li>Remove the frozen lentivirus from the -80 &#176;C freezer and thaw an aliquot on ice before each use; do not refreeze.</li>
	<li>While the virus is thawing, warm the cell culture medium containing the serum compatible with the cell line of interest. Once the virus is fully thawed, prepare a range of dilutions (1:3, 1:10, 1:30, and 1:100) in DMEM in a fresh 1.5 mL microfuge tube.</li>
	<li>Bring up the volume in the tubes to 1 mL with reduced serum media.</li>
	<li>Add 2 &#181;L of Polybrene (at a stock of 4 &#181;g/&#181;L) to 1 mL of virus/media. Mix well by pipetting and add 1 mL of mixture to the cells. Incubate the cells with the virus for 24 h.</li>
	<li>Remove the virus media and replace with normal DMEM media supplemented with 10% FBS. Maintain the cells at 37 &#176;C, 5% CO2.</li>
	<li>Monitor the growth of the cells and change the culture media every two days. At confluence, expand the 6-well dish into a 10-cm diameter tissue culture plate.</li>
	<li>Once the cells have been sufficiently expanded, seed 1.5 x 104 cells per well in 50 &#181;L of DMEM media on 384-well assay plates to check the expression level of the YFP tagged protein by microscopy. If the YFP tagged protein cell expression shows a signal-to-noise ratio &#8805;3, prepare cell stock for the corresponding cell line.</li>
</ol>
3. Time dependent effect of proteasome inhibitor on protein aggregation
NOTE: Perform image acquisition using an automated microscope (see Materials).
<ol>
	<li>Dispense 0.25 &#181;L of DMSO or proteasome inhibitor dissolved in 100% DMSO (ALLN, 2 mM) on 384-well flat bottom plates.</li>
	<li>Manually seed 1.5 x 104 cells per well in 50 &#181;L of DMEM media on the same assay plate. The proteasome inhibitor (ALLN) final concentration should be 10 &#181;M.</li>
	<li>Set up the microscope system environmental control unit to 37 &#176;C and 5% CO2. A screenshot of the experimental setup is shown in Figure 3.</li>
	<li>Operate the microscope in wide field fluorescence mode, using the software with the following settings: LWD 20X objective, non-confocal mode, 4 fields/well, with a capture interval of an hour. At the end of each run, captured images are automatically uploaded to the server.</li>
</ol>
4. Time lapse imaging for YFP expression in living cells, and image analysis (single channel)
NOTE: The following steps describe application of the software (e.g., Columbus).
<ol>
	<li>Select the appropriate algorithm to segment the primary objects (cytosol and aggregates). If required, adjust background threshold and contrast parameters (Figure 4).</li>
	<li>Count cells using &#39;find cells&#39; with an individual threshold of 0.1 for the YFP intensity channel. Determine aggregates using a &#39;find spots&#39; algorithm with a relative YFP spot intensity &#62;0.2 and a splitting coefficient of 1.0.</li>
</ol>
5. Dose response effect of proteasome inhibitors on protein aggregation on living cells stained for their nuclei (two channels)
NOTE: Perform image acquisition using an automated microscope. Determine the concentration range of proteasome inhibitors (ALLN, Epoxomicin, and MG132) based on the expected IC50 value to ensure an optimal curve fit.
<ol>
	<li>Prepare serial dilutions of compounds on a 384-well storage polypropylene plate by the standard 1:3 dilution series. Make sure to mix the compound dilutions well to ensure that the compound concentrations are accurate.</li>
	<li>Dispense 0.25 &#181;L of proteasome inhibitors on empty flat-bottom black 384-well assay plates. We use a liquid handler equipped with a 384-capillary head capable of transferring volumes.</li>
	<li>Seed 1.5 x 104 cells per well in 50 &#181;L of DMEM Media on assay plates. Incubate the assay plates at 37 &#176;C and 5% CO2 for 24 h.</li>
	<li>Add 10 &#181;L of DMEM Media (pre-warmed at 37 &#176;C) with staining solution (Hoechst 33342 at a stock of 10 mg/mL) and incubate at room temperature for 10 min.</li>
	<li>Launch the microscope operating software. A screenshot of the experimental setup is shown in Figure 6. Select the &#34;Configuration&#34; tab and select 20X&#160;objective and the correct plate type. Ensure &#34;collar&#34; is set to the correct value on the objective allowing proper focus with different plate types.</li>
	<li>Select the &#34;Microscope&#34; tab. Define exposure 1 as YFP (488 laser) and exposure 2 as Hoechst (405 laser). Activate the filter on both exposures and assign exposure 1 to camera 1 and exposure 2 to camera 2. Set exposure times to ~800 ms for exposure 1 and ~40 ms for exposure 2.</li>
	<li>Select exposure 1. Set focus height to 0 &#181;m. Select &#34;Focus&#34;. Once focused, expose camera 1. Adjust the focus height to optimize the exposure plane and click on &#34;Take height&#34;. Change the exposure times and laser power to give a maximum pixel intensity of ~3,000. Save exposure parameters. Repeat for exposure 2.</li>
	<li>Select &#34;Experiment Definition&#34; tab. Create a layout and sublayout. Drag and drop the relevant layout, exposure, reference image, skewcrop file, and sublayout. Save the experiment.</li>
	<li>Select &#34;Automatic Experiment&#34; tab and acquire images. At the end of each run, captured images are automatically uploaded to the server.</li>
</ol>
6. Image analysis of two channel images
<ol>
	<li>Select the software algorithm to segment the primary objects (nuclei, cytosol, and aggregates) (Figure 7).</li>
	<li>Select the method that segments nuclei accurately by visual inspection of the segmented objects. Hoechst-stained objects in channel 1 (Ch1) will be used to determine the number of cells. The YFP staining from the mutant SOD1-A4V in channel 2 (Ch2) will be used to determine the amount of aggregates.</li>
	<li>Count cells using the &#39;find nuclei&#39; algorithm as Hoechst-staining regions &#62;20 &#181;m2, with a split factor of 7.0, an individual threshold of 0.40, and a contrast &#62;0.10.</li>
	<li>Create the data table and determine EC50 for each of the compounds using the &#39;Non-linear regression&#39; equation in the graphing software.</li>
</ol>

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The authors have nothing to disclose.

There are two main approaches for generating stable cell lines. The first takes several weeks and requires transient transfection and resistance selection of the genomic integrated plasmid DNA vectors. The second takes a matter of hours through the use of lentivirus, making this protocol amenable to the effective expression of target protein in multiple cell lines with limited effort. The TRIP-CMV vector19 was a sustained vector to use, with conserved transduction efficiency and stable transgene e...

Protein aggregation is a biological process by which misfolded proteins group up and may act as causative agents in neurodegenerative diseases (amyloidosis). Characterizing protein aggregation is essential in understanding the role of aggregates in cellular dysfunction as well as in facilitating the discovery of new factors that influence the onset of the pathology. The visualization of fluorescence-tagged proteins in living cells is a powerful method which may aid in the development of assays applicable to high content screening (HCS)1,2,3,4.
Amyotrophic lateral sclerosis (ALS) is regarded as a proteopathic disease caused by the presence of misfolded proteins with the propensity to aggregate and accumulate in motor neurons in both familial ALS (fALS) and sporadic ALS (sALS)5,6. A subset of ~20% of fALS cases are associated with dominant mutations in the gene encoding the cytosolic antioxidant enzyme copper-zinc superoxide dismutase type 1 (SOD1)7,8. Several potential causes for this genetically derived dysfunction have been proposed, including changes in the structure and function of SOD1 variants, such as aberrant stability, increased unfolding rate, and propensity to aggregate9,10. Notably, the only verified and potentially toxic property shared by both ALS-linked SOD1 variants and wild-type (WT) SOD1 is an increased propensity to form compartmentalized protein aggregates or proteinaceous inclusions11,12. Misfolded mutant SOD1, is persistently polyubiquitinated and degraded by the ubiquitin-proteasome system. As a result, a low level of inhibition of proteasome activity leads to the accumulation of mutant SOD1 aggregates13,14, which form amorphous structures composed of soluble components that can exchange with soluble mutant SOD1 in the cytosol15. In particular, SOD1 mutant A4V (alanine at codon 4 changed to valine) is the most common ALS-causing mutation, and leads to rapid neurodegeneration with an average survival time of less than 2 years after disease onset16. Biochemically, SOD1 A4V has an increased tendency to monomerize, aggregate, and form amyloid pores; its pore-like aggregates are similar to amyloid pores of other disease-linked mutant forms, such as &#945;-synuclein and &#946;-amyloid protein17. To study the dynamics of SOD1-aggregate accumulation, methods for monitoring soluble and insoluble SOD1 aggregate forms remain to be developed.
We have previously shown, using live-cell imaging and HEK-293 cells transiently transfected with fluorescent protein-tagged SOD1, that ALS-associated mutations impair SOD1 dimerization and aggregation11. Although transient expression systems can provide useful information about the biological outcome of short-term gene overexpression, methods providing stable integration of desired genes may be preferred for assay development. As such, lentiviral vectors offer the ability to confer long-term and regulated gene expression on mammalian cells 18. In this study, we focused on the generation of stable cell lines transduced with recombinant lentivirus bearing WT and mutant SOD1 tagged with yellow fluorescent protein (YFP). Using live-cell imaging microscopy and automated quantification of SOD1 aggregation, we triggered and quantified SOD1 aggregation events upon inhibition of the proteasome.

<table>
	<tbody>
		<tr>
			<td>ALLN (C20H37N3O4)</td>
			<td>Millipore</td>
			<td>208719</td>
			<td></td>
		</tr>
		<tr>
			<td>MG132 (C26H41N3O5)</td>
			<td>Sigma-Aldrich</td>
			<td>C2211</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxomicin (C28H50N4O7)</td>
			<td>Sigma-Aldrich</td>
			<td>E3652</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H-3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera</td>
			<td>Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera EvoShell software</td>
			<td>Perkin Elmer</td>
			<td>Ver 1.8.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Operetta</td>
			<td>Perkin Elmer</td>
			<td>OPRT1288</td>
			<td></td>
		</tr>
		<tr>
			<td>Harmony Imaging software</td>
			<td>Perkin Elmer</td>
			<td>Ver 3.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Columbus Image analysis software</td>
			<td>Perkin Elmer</td>
			<td>Ver 2.3.2</td>
			<td></td>
		</tr>
		<tr>
			<td>CyBi Hummingwell liquid handling</td>
			<td>CyBio AG</td>
			<td>OL 3387 3 0110</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay development for high content quantification of sod1 mutant protein aggregate formation in living cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

Generating stable cell line using lentivirus: The overall strategy for monitoring SOD1 protein aggregates is illustrated in Figure 1. In a first step, we generated a lentiviral expression vector for SOD1 stable gene delivery into cell lines (Step 1). Two lentiviral vectors encoding YFP-tagged SOD1 WT and SOD1 A4V (SOD1WT-YFP and SOD1A4V-YFP, respectively) with packing and envelope plasmids were prepared (Figure 2A</strong...

Watch this Scientific Journal Video about Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells at JoVE.com

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

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Research

JoVE Journal

Neuroscience

7.1K Views.  Institut Pasteur Korea. We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Video: Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that 
initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The 
recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca2+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca2+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.
Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the 
photoisomerization of its 11-cis chromophore to all-trans5-7. This regeneration begins with the release of all-trans retinal by 
the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all-
trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is 
then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).
Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca2+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer 
segment Ca2+ changes and response to light8-12 as well as the role of inner segment Ca2+ stores in Ca2+ homeostasis13,14. 
Fluorescent dyes can also be used for measuring Mg2+ concentration15, pH, and as tracers of aqueous and membrane compartments16. 
Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single 
photoreceptor cells17-19.

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca2+ or other factors.

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.

We present an ex vivo cell migration assay that allows precise quantification of enteric neural crest cell migration potential in the presence of various growth factors.

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.

Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other neurological diseases at an increasing speed. However, little is known about the molecular mechanisms that underlie these disorders. The genetic, molecular, and behavioral toolbox of Drosophila melanogaster makes this model organism particularly useful to characterize new disease genes and mechanisms in a high-throughput manner. Nevertheless, high-throughput screens require efficient and reliable assays that, ideally, are cost-effective and allow for the automatized quantification of traits relevant to these disorders. The island assay is a cost-effective and easily set-up method to evaluate Drosophila locomotor behavior. In this assay, flies are thrown onto a platform from a fixed height. This induces an innate motor response that enables the flies to escape from the platform within seconds. At present, quantitative analyses of filmed island assays are done manually, which is a laborious undertaking, particularly when performing large screens.
This manuscript describes the &#34;Drosophila Island Assay&#34; and &#34;Island Assay Analysis&#34; algorithms for&#160;high-throughput, automated data processing and quantification of island assay data. In the setup, a simple webcam connected to a laptop collects an image series of the platform while the assay is performed. The &#34;Drosophila Island Assay&#34; algorithm developed for the open-source software Fiji processes these image series and quantifies, for each experimental condition, the number of flies on the platform over time. The &#34;Island Assay Analysis&#34; script, compatible with the free software R, was developed to automatically process the obtained data and to calculate whether treatments/genotypes are statistically different. This greatly improves the efficiency of the island assay and makes it a powerful readout for basic locomotion and flight behavior. It can thus be applied to large screens investigating fly locomotor ability, Drosophila models of movement disorders, and drug efficacy.

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

The island assay is a relatively new, cost-effective assay that can be used to evaluate the basic locomotor behavior of Drosophila melanogaster. This manuscript describes algorithms for automatic data processing and objective quantification of island assay data, making this assay a sensitive, high-throughput readout for large genetic or pharmacological screens.

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. Transcytosis across the BBB regulates the delivery of hormones, metabolites, and therapeutic antibodies to the brain parenchyma. Here, we present a protocol that combines immunofluorescence of free-floating sections with laser scanning confocal microscopy and image analysis to visualize subcellular organelles within endothelial cells at the BBB. Combining this data-set with 3D image analysis software allows for the semi-automated segmentation and quantification of capillary volume and surface area, as well as the number and intensity of intracellular organelles at the BBB. The detection of mouse endogenous immunoglobulin (IgG) within intracellular vesicles and their quantification at the BBB is used to illustrate the method. This protocol can potentially be applied to the investigation of the mechanisms controlling BBB transcytosis of different molecules in vivo.

Here we present a microscopy-based protocol for high-resolution imaging and a three-dimensional reconstruction of the mouse neurovascular unit and blood-brain barrier using brain free-floating sections. This method allows for the visualization, analysis, and quantification of intracellular organelles at the BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...