imaging of fluorescently-labeled motor neurons in an adult drosophila leg

Imaging of Fluorescently-Labeled Motor Neurons in an Adult Drosophila Leg

To image the legs, use the 488-nanometer laser and two detectors simultaneously to set up the first track for obtaining both the GFP and cuticle autofluorescence. Select a 20 to 25x oil immersion objective and set the resolution to 1024 by 1024 pixels, with a 12-bit depth. And set the z spacing to 1 micrometer. Load the slide onto the microscope stage.And using the same laser power for both detectors, adjust the gain of the first detector to obtain a bright GFP signal. And adjust the second detector to ensure that some areas with a high cuticle signal produce a saturated signal in this detector. Then, image the legs, using the tile or position options to capture the entire leg, if a leg is extended, or too large to be imaged in a single frame.For image processing, open the confocal stack in ImageJ Fiji. And use the formats plugin to open any images that are not in TIFF format. To split the channels, select Image, Color, and Split Channels. To subtract the cuticle signal from the GFP signal, open Process and Image Calculator, and select the stack from detector one as Image1.In the operation window, select Subtract, and select the track from Detector 2 as Image2, so that only the endogenous GFP signal will be obtained. Use Image, Stacks, Z Protect to generate max intensity projection for the endogenous GFP signal. Use the controls in the brightness control window to adjust the brightness and contrast.

imaging-fluorescently-labeled-motor-neurons-an-adult-drosophila

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ALL procedures are found to the tejlat elk1. Leg Dissection and Fixation<ol><li>Take a glass multi-well plate and fill the appropriate number of wells with 70% ethanol. Add 15&#8211;20 carbon-dioxide-anesthetized flies (of either sex and any age) to each well, and using a brush, gently dab the flies into the ethanol solution until they are fully submerged. NOTE: This step removes the hydrophobicity of the cuticle. Do not wash for more than 1 minute, because this increases the auto-fluorescence of the cuticle.</li><li>Rinse the flies three times with a 0.3% nonionic surfactant detergent solution in 1x phosphate-buffered saline (PBS). Keep the flies in this solution for at least 10 minutes. NOTE: Legs are better fixed when detergent is included, as this is likely to increase the fixative's penetration inside the leg.</li><li>Place a multi-well plate on the ice along with a tube of 4% paraformaldehyde (PFA). NOTE:&#160;It is critical to prepare fresh 4% PFA from a 16% PFA ethanol-free stock solution.</li><li>Use forceps to remove the head and abdomen of the flies without damaging the thoracic segment or the legs. NOTE: Removing the abdomen makes it easier to hold the fly and dissect the legs.</li><li>Dissect the legs from the thoracic segment with forceps and place them in the wells containing 4% PFA. Using the tip of fine forceps, push gently but firmly at the coxa-thorax junction until the leg detaches.</li><li>Fix the legs in 4% PFA overnight at 4 &#176;C (approximately 20 h total).</li><li>Wash the legs with 0.3% nonionic surfactant detergent solution in 1x PBS, 5x for 20 min each.</li><li>Replace the washing buffer with a mounting medium. Keep the leg in the medium for at least a day before mounting to allow for its complete penetration into the leg. NOTE: If the mounting medium is highly viscous, dilute it to 80% with 1x PBS because a sudden change in viscosity can cause the cuticle to collapse and damage the overall structure of the leg. Depending on the Gal4 driver, flies can be preserved for 1 to 3 weeks at 4 &#176;C in mounting media until mounting.</li></ol>2. Leg Mounting<ol><li>Place approximately 20 &#181;L of 70% glycerol on the left-hand side of a microscope slide and cover with a square 22 x 22 mm2 coverslip (Figure 1A, B). Pipette about 10 &#181;L of mounting medium along a line parallel to and at a small distance from the right edge of this coverslip. Add another 30 &#181;L of mounting medium further on the right side of the slide (Figure 1C). NOTE: When applying a coverslip over the legs (see below), the mounting medium will gently spread under the coverslip and around the legs without displacing them.</li><li>Using fine forceps, lift the leg from the solution and gently put it on the mounting medium near the left coverslip. Do the same for each leg and align them from top to bottom (Figure 1D).<ol><li>Lift and transfer the legs in a drop of medium held between both tips of the forceps. Orient the legs in two ways: external side up or down.</li></ol></li><li>Once all the legs (up to 6&#8211;8 legs can be mounted) are properly aligned, put a second coverslip over the legs such that this coverslip rests slightly on the previously placed coverslip&#160;(Figure 1E) to allow for space between the coverslip and the tissue and to prevent the legs from getting damaged (Figure 1G). NOTE: Alternatively, use sticker wells or orthodontic wax to create space between the coverslip and the slide.</li><li>Use a nail polish to secure the position of the coverslips at each corner (Figure 1F). NOTE: The tissue is now ready to image.</li></ol>3. Imaging<ol><li>Set up a single track using the 488 nm laser and two detectors simultaneously to obtain the GFP fluorescence and cuticle auto-fluorescence. Using the range control or the slider display in the spectral window of the light path window of the software, set the detection range of one detector (detector 1) between 498&#8211;535 nm and that of the other (detector 2) between 566-652 nm. NOTE: Typically, detector one should be a GaAsP detector, and detector two should be a photomultiplier tube. The 498-535 channel optimally detects the GFP signal but also detects autofluorescence from the cuticle. However, the 566&#8211;652 channel detects only the autofluorescence from the cuticle (Figure 2A).</li><li>Use a 20X or 25X oil immersion objective, 1024 x 1024 pixels resolution, 12-bit depth, and set Z spacing as 1 &#181;m. Set pixel dwell time as approximately 1.58 &#181;s, and average two frames/image. Use objectives with high numerical aperture.</li><li>Set all other settings, depending on the confocal microscope and imaging software used.<ol><li>Make sure to obtain a brighter cuticle signal from the 566&#8211;652 detector rather than from the 498&#8211;535 detector. To do this, use the same laser power of the 488-laser for both detectors. Using the saturation markers, first adjust the gain of detector 1 to obtain a bright enough GFP signal (Figure 2B, D). Then, adjust the gain of detector 2 to ensure that some areas with high cuticle signal (edges of legs, joints) produce a saturated signal in this detector (Figure 2C, E).</li><li>If the leg is extended and too large to be imaged in a single frame, use the Tile or Position option from the microscope imaging software.</li></ol></li><li>Reconstruct the final images either using the proprietary microscope imaging software or using ImageJ/FIJI freeware (see below).</li></ol>4. Post Imaging Processing<ol><li>Open the confocal stack in ImageJ/FIJI.<ol><li>Use the Bioformats plugin (Plugins| Bio-Formats| Bio-formats Importer) in FIJI to open images that are not in .TIF format from proprietary imaging software packages.</li></ol></li><li>Split the channels by selecting Image| Color| Split Channels. NOTE: If images from several positions were made and must be recombined, use the pairwise stitching plugin in FIJI from (Plugins &gt; Stitching &gt; Pairwise Stitching).</li><li>To subtract the cuticle signal from the GFP signal, open the Image Calculator (Process| Image Calculator): select the stack from detector one as Image 1 (GFP + cuticle) (Figure 3A), on the operation window, select subtract, and select the stack from detector two as Image 2 (cuticle) (Figure 3B). As a result, only the endogenous GFP signal (GFP) is obtained (Figure 3C).<ol><li>Merge back this stack (GFP) with the &#8216;cuticle-only&#8217; stack obtained from detector 2 (Figure 3B Image| Color| Merge Channels) to obtain an RGB image comprising of the tissue-specific GFP signal as well as the cuticle signal, which helps identify the axon arbors within the leg segments (Figure 3D).</li><li>Adjust the contrast and brightness of each image (Image| Adjust| Brightness/Contrast) and then generate a single RGB image (Image| Type| RGB Color). NOTE: To generate a maximum projection of the Z-stack (Image| Stacks| Z Project&#8230;), use maximum intensity projection for the GFP signal and average intensity for the cuticle, which can then be adjusted for brightness prior to merging the two channels.</li></ol></li><li>Alternatively, use a macro for automatic subtraction and processing of images in ImageJ/FIJI. Copy the text in Supplemental File 1 in the macro editor (Plugins| New| Macro), save the macro in the Plugins folder, and restart ImageJ/FIJI once to be able to use the macro.<ol><li>To use the macro, open the confocal stack and open the Brightness/Contrast window (Image| Adjust| Brightness/Contrast). Then, run the macro (Plugin| Macro| Run) and follow the instructions.</li><li>When asked to specify the operation (Image calculator window), choose Image 1 as stack 1 (GFP), Image 2 as stack 2 (cuticle), and Subtract. NOTE: The macro generates a maximal projection image of the processed GFP signal (MAX_Result of stack1), an average projection of the cuticle (AVG_stack2), the result of the subtraction of the cuticle stack from the GFP stack (Result of stack1), and the unprocessed cuticle stack (stack2).</li><li>When asked to adjust contrast, use the controls in the brightness/control window to adjust the brightness/contrast of the maximal projection image of GFP and of the average projection of the cuticle to generate an RGB merged image of both signals showing the GFP in green and the cuticle in gray. Merge Results of stack1 and stack2 to similarly generate a combined GFP and cuticle stack that can be used in the next stage.</li></ol></li><li>To visualize the legs in three dimensions, use specialized 3D software with the newly generated stacks (Figure 3E).<ol><li>Open the RGB stack with 3D software (see&#160;Table of Materials). In the pop-up dialog window, select all channels in the mode conversion section and enter the size of a voxel (volume of a pixel). NOTE: All necessary information is contained in the image files from whatever proprietary microscope software being used.</li><li>On the channel 2 module (GFP signal), right-click and select Display | volren. Then, left-click on the volren module. In the properties section (bottom left of the screen), click left on edit and select volrengreen.col. On the channel 1 module (cuticle signal) right-click and select Display | volren. Then, left-click on the volren module. In the Properties section, left-click on advanced and select DDR (a transparent radiograph-like display), then adjust the gamma value to see the cuticle background (Figure 3E).</li><li>To generate a 3D rotation, right-click on the project stack module. Then select animate| rotate. Left-click on the rotate module.</li><li>In the properties section, click on use bbox center. On the rotate module, right-click and select compute| movie maker. On the movie maker module, click the left mouse button. In the properties section, click left on Advanced (at the top right of the properties section).</li><li>In the file name field, fill in the name of the movie rotation. In the quality field, write 1 (max quality). Leave the frame at 200 if an 8.3 s rotation is desired (this number can be decreased or increased to modify the speed of the rotation). Then press apply (green button, bottom left of the properties section&#160;</li></ol></li></ol>

<figure class='table'><table class='ck-table-resized' style='border-collapse:collapse;font-family:Calibri;font-size:11pt;table-layout:fixed;' cellspacing='0' cellpadding='0' dir='ltr' border='1' data-sheets-root='1' data-sheets-baot='1'><colgroup><col style='width:25%;' width='265'><col style='width:25%;' width='248'><col style='width:25%;' width='216'><col style='width:25%;' width='293'></colgroup><tbody><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Ethanol absolute</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Fisher</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>E/6550DF/17</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Absolute analytical reagent grade</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Nonionic surfactant detergent</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Sigma-Aldrich</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>T8787</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Triton X-100, for molecular biology</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Fine forceps</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Sigma-Aldrich</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>F6521</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Jewelers forceps, Dumont No. 5</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Glass multi-well plate</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Electron Microscopy Sciences</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>71563-01</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>9 cavity Pyrex, 100x85 mm</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Paraformaldehyde (PFA)</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Thermofisher</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>28908</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Pierc 16% Formaldehyde (w/v), Methanol-free</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Glycerol</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Fisher BioReagents</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>BP 229-1</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Glycerol (Molecular Biology)</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Spacers</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Sun Jin Lab Co</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>IS006</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>iSpacer, four wells, around 12 &#956;L working volume per well, 7 mm diameter, 0.18 mm deep</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Square 22x22 mm coverslips</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Fisher Scientific</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>FIS#12-541-B</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>No.1.5 -0.16 to 0.19mm thick</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Mounting Medium</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Vector Laboratories</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>H-1000</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Vectashield Antifade Mounting Medium</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Confocal microscope</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Carl Zeiss</td><td style='overflow:hidden;padding:0px 3px;vertical-align:bottom;'>&#160;</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>LSM780; objective used LD LCI Plan-Apochromat 25x/0,8 Imm Korr DIC M27 (oil/ silicon/glycerol/water immersion) (420852-9871-000)</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Imaging software</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Carl Zeiss</td><td style='overflow:hidden;padding:0px 3px;vertical-align:bottom;'>&#160;</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>ZEN 2011</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>3D-Image software</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>ThermoFisher Scientific</td><td style='overflow:hidden;padding:0px 3px;vertical-align:bottom;'>&#160;</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>Amira 6.4</td></tr><tr style='height:20px;'><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>ImageJ</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>National Institutes of Health</td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'><a class='in-cell-link' href='https://imagej.nih.gov/ij/' target='_blank'>https://imagej.nih.gov/ij/</a></td><td style='color:#1a202c;overflow:hidden;padding:0px 3px;vertical-align:bottom;white-space:normal;word-wrap:break-word;wrap-strategy:4;'>ImageJ/FIJI</td></tr></tbody></table></figure>

Source:&#160;<a style='text-decoration:none;' href='https://appjove.unicartagenaproxy.elogim.com/v/58365/visualize-drosophila-leg-motor-neuron-axons-through-the-adult-cuticle'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Guan, W.,&#160;et. al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Visualize&#160;Drosophila&#160;<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Leg Motor Neuron Axons Through the Adult Cuticle.&#160;J. Vis. Exp<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. (2018)</a> &#160;<meta charset='utf-8'>This video demonstrates imaging of&#160;Drosophila legs with green fluorescent protein-expressing motor neurons using confocal microscopy. A laser and two detectors capture GFP and cuticle autofluorescence. Images are processed by splitting channels, subtracting autofluorescence, and merging into a colored composite image to visualize axons within the leg segment.

Source:&#160;Guan, W.,&#160;et. al. Visualize&#160;Drosophila&#160;Leg Motor Neuron Axons Through the Adult Cuticle.&#160;J. Vis. Exp. (2018)&#160;This ...

Begin with a confocal microscope setup containing a fixed, genetically modified Drosophila leg mounted within the space between two coverslips. The motor neuron axons in the leg express a membrane-tagged fluorescent protein.Use a single laser and two detectors to capture fluorescent signals from the motor neuron axons and background autofluorescence of the cuticle which is the outermost layer of the leg.Adjust imaging settings for optimal resolution and image quality.Fine-tune the brightness to ensure a bright axonal signal and a saturated cuticle autofluorescence.Capture an image with both axonal signal and cuticle autofluorescence.Using appropriate image processing software, open the captured image.Split the channels and subtract the background cuticle autofluorescence from the axonal signal.Adjust the brightness and contrast of both signals to improve image clarity.Merge the channels to generate a merged image to visualize the motor neuron axons within the leg segment.

16 Views. Imaging of Fluorescently-Labeled Motor Neurons in an Adult Drosophila Leg

Video: Imaging of Fluorescently-Labeled Motor Neurons in an Adult Drosophila Leg - Experiment

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

JoVE Journal

Developmental Biology

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Imaging of Fluorescently-Labeled Motor Neurons in an Adult Drosophila Leg

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Imaging of Fluorescently-Labeled Motor Neurons in an Adult Drosophila Leg