Name: application of multicolor flpout technique to study high resolution single cell morphologies and cell interactions of glia in drosophila
Uploaded: 2017-10-20T14:00:00
Description: Watch this Scientific Journal Video about Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila at JoVE.com

The authors thank Arnim Jenett, Aljoscha Nern, and other members of the Rubin laboratory for advice and sharing of unpublished reagents and the Janelia Fly Light Project Team for generating confocal images. The authors also thank the members of the Gaul laboratory for comments on the manuscript.

1. Preparing Flies for MultiColor FlpOut (MCFO) Experiments
NOTE: The MCFO technique refers to a modified version of the so called Flp-mediated stop cassette excision (FlpOut). Transgenic MCFO flies carry a heat shock promoter (hsp)-Flp recombinase and different reporters under UAS control. Each reporter consists of a common backbone of a myristoylated (myr) super folder green fluorescent protein (sfGFP), in which 10 copies of an epitope tag (e.g., HA, FLAG or V5) have been inserted. Th...

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This protocol describes an easy and efficient method to study the morphology of different cell types within a tissue of interest at high resolution. With the MCFO technique, multiple reporters with different epitope tags are used in combination for multicolor stochastic labeling (Figure 2). Similar to other methods such as Brainbow/Flybow15,16,17, MCFO increases the label diversity through marker co...

Glia, the non-neuronal cell population of the nervous system (NS), were long believed to provide a static framework for neurons and therefore were not studied in detail. However, in humans, glia constitute the vast majority of cells in the NS (~90%) and fall into several different categories, including astrocytes, oligodendrocytes, microglia and Schwann cells. In Drosophila, glia constitute about 10% of the cells in the NS. Intriguingly, their morphologies and functions are remarkably similar to those found in vertebrates1,2. Their morphologies include blood-brain barrier (BBB) forming epithelia, ensheathing, and astrocyte-like cells.
The Drosophila central nervous system (CNS) consists of the following principal structures: cortex regions that contain the neuronal cell bodies; neuropils that harbor synaptic connections; small and large axon tracts that connect the different neuropiles; peripheral nerves that connect sensory organs and muscles with the CNS (Figure 1). Glia are found associated with all these anatomical structures: Cortex glia (CG) in the cortical regions, astrocyte-like glia (ALG) and ensheathing glia (EG) in the neuropile regions, ensheathing glia are also associated with central axon tracts and peripheral nerves (EGN), and finally, two sheet-like glia, perineurial glia (PG) and subperineurial (SPG), which together form a contiguous layer that covers the entire NS (Figure 2).
Previous studies have shown that glia play important roles in the development of the NS; they monitor neuronal cell numbers by reacting to systemically circulating insulin-like peptides, provide trophic support to neurons, such as the astrocyte-neuron lactate shuttle, and eliminate dying neurons by phagocytosis3,4,5,6. In the mature NS, glia maintain the BBB, take up neurotransmitters and maintain ionic homeostasis, act as the major immune cells in the NS, since macrophages cannot breach the BBB, and modulate synaptic activity as well as animal behavior6,7,8,9,10,11.
Whether the different glial subtypes perform specialized functions remains an important open question. However, a systematic genome-wide analysis of glia, especially in the adult, has been hampered by a lack of appropriate genetic tools for their manipulation. Here, a method that allows the efficient and easy characterization of cell shapes to study complex cell-cell interactions is presented. This technique has been applied to characterize the morphology of the different glial subtypes in the adult Drosophila brain, but, depending on the specific GAL4 driver used, it could be adapted to study neurons12,13, any kind of intermingling cells, and in principle any tissue in any developmental stages.

<table>
	<tbody>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>GD100</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Sarstedt</td>
			<td>72.737.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting dish 30 mm x 12 mmm</td>
			<td>Electron Microscopy Sciences</td>
			<td>70543-30</td>
			<td>Glass dissection dish</td>
		</tr>
		<tr>
			<td>Pyrex 3 Depression Glass Spot Plate</td>
			<td>Corning</td>
			<td>7223-34</td>
			<td>Glass dissection plates</td>
		</tr>
		<tr>
			<td>Sylgard Black</td>
			<td>SYLGARD, Sigma-Aldrich</td>
			<td>805998</td>
			<td>home made with charcoal</td>
		</tr>
		<tr>
			<td>ExpressFive S2 cell culture medium</td>
			<td>Invitrogen</td>
			<td>10486-025</td>
			<td></td>
		</tr>
		<tr>
			<td>20% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Roth</td>
			<td>3051.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson Laboratories</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson Laboratories</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit HA-tag</td>
			<td>Cell Signaling</td>
			<td>C29F4</td>
			<td>Primary antibody, dilution 1:500</td>
		</tr>
		<tr>
			<td>Rat FLAG-tag</td>
			<td>Novus Biologicals</td>
			<td>NBP1-06712</td>
			<td>Primary antibody, dilution 1:100</td>
		</tr>
		<tr>
			<td>Mouse V5-tag:DyLight 549</td>
			<td>AdSerotec</td>
			<td>0411</td>
			<td>Conjugated antibody, dilution 1:200</td>
		</tr>
		<tr>
			<td>anti-rabbit AlexaFluor 488</td>
			<td>Invitrogen</td>
			<td>A11034</td>
			<td>Secondary antibody, dilution 1:250</td>
		</tr>
		<tr>
			<td>anti-rat DyLight 647</td>
			<td>Jackson Laboratories</td>
			<td>712-605-153</td>
			<td>Secondary antibody, dilution 1:100</td>
		</tr>
		<tr>
			<td>Vecta Shield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFate Gold</td>
			<td>Invitrogen</td>
			<td>S36937</td>
			<td></td>
		</tr>
		<tr>
			<td>Secure Seal Spacer</td>
			<td>Grace Biolabs</td>
			<td></td>
			<td>Contact company for ordering</td>
		</tr>
		<tr>
			<td>Microscope cover glass 22 X 60 mm</td>
			<td>Marienfeld</td>
			<td>101152</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass 22 x 22 mm</td>
			<td>Roth</td>
			<td>H874</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope, Leica MZ6</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope LSM710</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol</td>
			<td>Zeiss</td>
			<td>518 F</td>
			<td>Immersion oil for fluorescence-microscopy, halogen free</td>
		</tr>
		<tr>
			<td>Immersol</td>
			<td>Zeiss</td>
			<td>W 2010</td>
			<td>Immersion fluid for water-immersion objectives, halogen free</td>
		</tr>
		<tr>
			<td>R56F03-GAL4 (EG)</td>
			<td>Bloomington Stock Center</td>
			<td>39157</td>
			<td>GAL4 driver</td>
		</tr>
		<tr>
			<td>R86E01-GAL4 (ALG)</td>
			<td>Bloomington Stock Center</td>
			<td>45914</td>
			<td>GAL4 driver</td>
		</tr>
		<tr>
			<td>hspFlpPestOpt; UAS-FRT-stop-FRT-myr-smGFP-HA, UAS-FRT-stop-FRT-myr-smGFP-FLAG, UAS-FRT-stop-FRT-myr-smGFP-V5</td>
			<td>Bloomington Stock Center</td>
			<td>64085</td>
			<td>UAS reporter (https://bdscweb.webtest.iu.edu/stock/misc/mcfo.php)</td>
		</tr>
		<tr>
			<td>Fiji (Image J)</td>
			<td></td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Multi Time Macro</td>
			<td>Zeiss</td>
			<td></td>
			<td>Software for automated scanning</td>
		</tr>
	</tbody>
</table>

application of multicolor flpout technique to study high resolution single cell morphologies and cell interactions of glia in drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

This section illustrates examples of results that can be obtained by using the MCFO technique in the adult Drosophila brain. Figure 3 shows a schematic of the method. Three differently membrane-tagged reporters (myr-smGFP-HA, myr-smGFP-FLAG and myr-smGFP-V5) under UAS control are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of Flp recombinase which rand...

Watch this Scientific Journal Video about Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila at JoVE.com

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

The overall goal of this technique is to visualize single cell morphologies in complex anatomical tissues, thus simplifying the study of cell cell interaction in Drosophila. This method can help to answer key questions regarding complex morphologies and cell cell interactions using the fruit fly. The main advantage of this technique is that it can be adapted to the study of single cell morphologies in any tissue and at any developmental stage.Demonstrating the procedure will be Anja Kieser, a technician in our laboratory. The MCFO technique modifies the standard FlpOut technique as follows. A heat shock FLP recombinase and different reporters remain under UAS control, however, each reporter has a common backbone of a myristoylated superfolder GFP, in which copies of an epitope tag have been inserted.The resulting non-fluorescent proteins are named spaghetti monster GFPs and are identified with antibodies. Depending on the number of excisions, a different combination of epitopes gets expressed. Because the HSP promoter is slightly active at 25 degrees Celsius, set up crosses at 18 degrees Celsius where it is truly inactive.Then, wait around 20 days to obtain the F1 generation. Sort the F1 females and males into separate vials. To heat shock them, first transfer them into fresh food vials when they are three to four days old.Younger flies can be too delicate to heat shock. Next, transfer the vials into a 37 degree Celsius water bath. Submerge them deeply to ensure a homogenous heat shock.For sparse labeling of glial cells, start with five to eight minutes of heat shock and then optimize. The optimal heat shock time will sparsely label the target cells and its relation depends on the specific GAL4 driver in use. After the heat shock, lay the vials horizontally on the bench for a few minutes to cool them down.Now, start maintaining the flies at 25 degrees Celsius in the same vials. After two days, reporter expression will be optimal and the flies can be dissected. In preparation, put three deep depression wells on ice.Fill one well with 70%ethanol, one with PBS and one with S2 cell culture medium. Then, load a three centimeter dissecting dish lined with black silicone with cold S2.Conduct the entire dissections in S2 to keep the tissues healthy. Now, anesthetize the flies with carbon dioxide.Then, using forceps or a feather, put the flies in cold 70%ethanol for about 30 seconds followed by cold PBS for about 30 seconds and then transfer them into cold S2.Thus, wash all the flies to be dissected. Up to 10 flies of one genotype is usually enough. Now, within 30 minutes, dissect out all the brains.First, detach the head from the rest of the body and discard the body. Keep the head held with the off hand through the entire dissection or it will be very difficult to regain a grip on it. Next, pull out one eye by gripping the tissue only just below the retina.Then pull off the other eye and thus expose the brain and surrounding tissues. Next, remove all the tracheal tissue and remaining cuticle around the brain, until the brain tissue appears clean. All the tracheal tissue must be removed for optimal staining results.This is all that is needed to prepare the brain for fixation. Proceed with dissecting all the brains, while keeping the dissected brains in cold S2.Try to have up to 10 brains prepared per microcentrifuge tube for optimal staining. Transfer the isolated brains using a P10 pipette tip into a 200 microliter microcentrifuge tube with fixative solution.Never handle the brains using forceps. Incubate the tissues protected from light. Avoid aspirating the brains during solution transfers.To remove the fixative, use three or more 15 minute washes with washing solution. Then block the tissues with blocking solution for 30 minutes or longer. Next, replace the blocking solution with primary antibodies diluted in wash solution and incubate the brains overnight at four degrees Celsius.To remove the primary antibodies, use three one hour washes in wash solution. Next, add secondary fluorophore conjugated antibodies in wash solution and incubate the brains overnight at four degrees Celsius or for four hours at room temperature. To completely wash off the unbound antibodies, use three one hour washes in wash solution followed by a longer wash with PBS.Then mount the brains. Prepare two cover slips with an imagining spacer. In the spacer and on the extra cover slip, apply 10 microliters of mounting medium with an anti-fade agent.Next, using a P10 pipette, transfer the brains to the cover slip, depositing them next to the medium along with some PBS. Do not let the tissue dry out. Now, carefully move the brains into the mounting medium using the pipette and finally move them to the medium in the spacer and arrange them with forceps.Then, remove the adhesive liner on the spacers and attach a cover slip. Secure the cover slip with a little pressure using forceps and proceed immediately with imaging or store the slides at minus 20 degrees Celsius for later analysis. Using the described methods, three different membrane tagged reporters are kept silent by transcriptional terminators flanked by FRT sites.When the animals were heat shocked, the FLP recombinase was expressed and randomly removed the terminators, leading to the expression of different combinations of reporters. Overall, the different reporter combinations provide seven different colors, thus multiple single cell morphologies can be visualized and studied individually. Various heat shock times were tested with EG-GAL4 and ALG-GAL4.The single cell labeling was analyzed in the antennal lobe region of the brain. The EG-GAL4 driver expressed in ensheathing glial cells that all ensheathed on the surface of the antennal lobe. By comparison, astrocyte-like glia targeted by the ALG-GAL4, covered mostly non-overlapping areas of the antennal lobe.After watching this video, you should have a good understanding of how to label single cells in solution. In order to understand their complex and anatomical relationships using Drosophila and in principle and in model organism in which the binary system GAL4 UIS may be applied. While attempting this procedure for the first time, it's important to remember first to optimize the heat shock time and to follow as much as possible the single steps of the staining protocol in order to obtain the best results.

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Research

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Behavior

Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.

The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant.  The NICU Network Neurobehavioral Scale ...

Study Motor Skill Learning by Single-pellet Reaching Tasks in Mice

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and retrieve food rewards positioned at a specific location, their motor performance (defined as accuracy and speed) improves progressively over time, and plateaus after persistent training. Once such reaching skill is mastered, its further maintenance does not require constant practice. Here we introduce a single-pellet reaching task to study the acquisition and maintenance of skilled forelimb movements in mice. In this video, we first describe the behaviors of mice that are commonly encountered in this learning and memory paradigm, and then discuss how to categorize these behaviors and quantify the observed results. Combined with mouse genetics, this paradigm can be utilized as a behavioral platform to explore the anatomical underpinnings, physiological properties, and molecular mechanisms of learning and memory.

Persistent practice improves the precision of coordinated movements.&#160;Here we introduce a single-pellet reaching task, which is designed to assess the learning and memory of forelimb skill in mice.&#160;

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

Tickling, a Technique for Inducing Positive Affect When Handling Rats

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk for both the animals and the hands of their handlers, decreased animal welfare, and less valid research data. To minimize negative effects on experimental results and human-animal relationships, research animals are often habituated to being handled. However, the methods of habituation are highly variable and often of limited effectiveness. More potently, it is possible for humans to mimic aspects of the animals&#39; playful rough-and-tumble behavior during handling. When applied to laboratory rats in a systematic manner, this playful handling, referred to as tickling, consistently gives rise to positive behavioral responses. This article provides a detailed description of a standardized rat tickling technique. This method can contribute to future investigations into positive affective states in animals, make it easier to handle rats for common husbandry activities such as cage changing or medical/research procedures such as injection, and be implemented as a source of social enrichment. It is concluded that this method can be used to efficiently and practicably reduce rats&#39; fearfulness of humans and improve their welfare, as well as reliably model positive affective states.

This article demonstrates the standardized application of playful handling, a tickling technique designed to mimic rat rough-and-tumble play. This technique is effective at reducing fearful reactions to humans and generating positive affect when rats are handled for common husbandry activities and medical and research procedures such as injection.

Handling small animals such as rats can lead to several adverse effects. These include the fear of humans, resistance to handling, increased injury risk ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...

Radiotracer Administration for High Temporal Resolution Positron Emission Tomography of the Human Brain: Application to FDG-fPET

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose analogue, 18F-fluordeoxyglucose (FDG-fPET), it is now possible to measure the dynamics of glucose metabolism with temporal resolutions approaching those of functional magnetic resonance imaging (fMRI). This direct measure of glucose uptake has enormous potential for understanding normal and abnormal brain function and probing the effects of metabolic and neurodegenerative diseases. Further, new advances in hybrid MR-PET hardware make it possible to capture fluctuations in glucose and blood oxygenation simultaneously using fMRI and FDG-fPET.
The temporal resolution and signal-to-noise of the FDG-fPET images is critically dependent upon the administration of the radiotracer. This work presents two alternative continuous infusion protocols and compares them to a traditional bolus approach. It presents a method for acquiring blood samples, time-locking PET, MRI, experimental stimulus, and administering the non-traditional tracer delivery. Using a visual stimulus, the protocol results show cortical maps of the glucose-response to external stimuli on an individual level with a temporal resolution of 16 s.

This manuscript describes two radiotracer administration protocols for FDG-PET (constant infusion and bolus plus infusion) and compares them to bolus administration. Temporal resolutions of 16 s are achievable using these protocols.

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose ...

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human populations, the Drosophila model allows strict control over genetic background, and virtually unlimited numbers of individuals of the same genotype can be reared rapidly under well-controlled environmental conditions without regulatory restrictions and at relatively low cost. Flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. Here, we describe a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The assay is based on video recording of single flies introduced without anesthesia in 24-well cell culture plates in a set-up that enables synchronous initiation of alcohol exposure. The system enables a single person to collect individual ethanol sedation data on as many as 2,000 flies within an 8 h work period. The assay can, in principle, be extended to assess the effects of exposure to any volatile substance and applied to measure effects of acute toxicity of volatiles on other insects, including other fly species.

High-Throughput Method for Measuring Alcohol Sedation Time of Individual Drosophila melanogaster

Current methods to measure alcohol sensitivity in Drosophila are designed to test groups of flies. We present a simple, low-cost, high-throughput assay for assessing alcohol sedation sensitivity in large numbers of single flies. The method does not require specialized tools and can be performed in any laboratory using common materials.

Drosophila melanogaster provides an excellent model to study the genetic underpinnings of alcohol sensitivity. In contrast to studies in human ...

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but effective method to observe and document the behavior or morphology of flies. Flies were placed in a translucent observation chamber c.a. &#216;15 x 5mm (no food inside) or &#216;15 x 12 mm (with an 8 mm-high piece of food inside). After covering with an ultraviolet (UV)/clear filter with high light transmittance, the chamber was placed under a 5-50x zoom stereo microscope, and mini light-emitting diode (LED) video lights were placed on both sides of the microscope to illuminate the chamber to obtain uniform, soft, bright, and nearly shadow-free light. Then, a compact digital camera with 3-5x optical zoom, which can record 1080 P high-definition or higher resolution video (at a frame rate of &#8805;30 fps), was connected to the eyepiece of microscope through a bracket, and photographs or videos were taken through the eyepiece. By adjusting the zoom knob of the zoom stereo microscope, it was very easy to track the flies and take panoramic or detailed close-up images as needed, while the camera recorded everything under the microscope. Because the flies can stay at any position in the chamber, they can be observed and recorded from all directions. The photographs or videos taken are of good image quality. This method can be used both for scientific research and teaching.

An Adjustable High-Definition Imaging System for Behavioral Studies of Drosophila Adults

This protocol describes how to make a simple adult Drosophila behavior observation chamber, and how to take high-definition photographs/videos of the morphology or behavior of different types of fruit flies in the observation chamber through relatively simple and affordable methods.

Drosophila melanogaster is a very powerful model in biological research, but a bad model for photography or videography. This paper describes a simple but ...