We thank Jean-Baptiste Sibarita and Daniel Choquet (IINS, CNRS/University of Bordeaux) for their kind support with the single-molecule analysis. We especially thank Nick Valmas and Jessica Mcgaw (QBI, University of Queensland) for their help with the video recording, voice-over as well as figure graphic design. This work was supported by the Australian Research Council (AR) discovery project (DP170100125 to F.A.M.), the Australian Research Council (LIEF Grant LE0882864 to F.A.M.), Future Fellowship (FT100100725 to B.V.S.), Australian Research Council (LIEF Grant LE130100078 to F.A.M.) and NHMRC Project grant (APP1103923 to B.V.S.). F.A.M. is a NHMRC Senior Research Fellow (ONT1060075).

1. Design of Transgenic Drosophila Expressing mEos2-Tagged Proteins
<ol>
	<li>Select the protein to be tagged with the mEos2 protein. To increase the imaging success rate, select proteins with a transmembrane domain in the neuronal plasma membrane14 (e.g., Syntaxin-1A), proteins on synaptic vesicles or autophagosomes26,27 (e.g., Synaptobrevin-2), or cytosolic proteins with close interaction with neuronal plasma membrane proteins (e.g., Munc18-1).</li>
	<li>Construct transgenes encoding the mEos2-tagged protein (Figure 1). Design the forward and reverse primers for the protein and mEos2. 
	NOTE: The mEos2 coding sequence can be amplified from the pmEos2-N1 plasmid, which is designed to fuse to the C-terminus of the protein of interest. Cloning can for example be performed by in vivo recombination in Saccharomyces cerevisiae using the pFL44S-attB-MCS-w+ vector14,28, alternately other cloning methods could be used such as Gibson assembly protocol29.
	<ol>
		<li>Linearize the vector with BamHI and XbaI and co-transform into ura3- yeast cells together with the PCR fragments encoding mEos2 and the protein of interest. Inject Drosophila embryos with the construct to generate a transgenic fly line30.</li>
	</ol>
	</li>
	<li>Rear the transgenic fly cultures on standard yeast and molasses medium or some other suitable substitute contained in plastic vials. To ensure healthy and fairly large synaptic boutons31, avoid overcrowding flies in the vials, and flip flies to new vials after each day of egg-laying; this ensures the third instar larvae are well fed and reach proper size by the time they commence wandering on the medium. Raise the transgenic fly at room temperature (25 &#176;C). 
	NOTE: Larger synaptic boutons tend to yield larger quantity of proteins visualized during imaging.</li>
</ol>
2. Dissection of Third Instar Larva
<ol>
	<li>Make a cylindrical or semicylindrical shaped polydimethylsiloxane (PDMS) base &#8804;20 mm in diameter and &#8804;20 mm in height using an appropriate silicone elastomer kit (Figure 2A). Mix silicone elastomer with a silicone hardening agent at a ratio of 10:1.
	<ol>
		<li>Stir the mixture thoroughly for 5 min. Pour the mixture into a mold with the dimsensions listed above. Let it harden in an oven at 100 &#176;C for 45 min. The PDMS base will serve as the foundation on which to carry out dissection. Ensure the PDMS base fits into the well of a glass-bottomed culture dish.</li>
	</ol>
	</li>
	<li>Select a wandering third instar larva from the vial wall. Use an optical stereo-microscope at 4.5 magnification to visualize the larva placed on the PDMS cylindrical base with the dorsal side up.</li>
	<li>Use curved and angled tweezers to stick minutien pins on the head over the mouth hooks, between the two extending anterior spiracles, and on the tail over the anus, between the two posterior spiracles. Add 40 &#181;L of Schneider&#39;s insect medium at room temperature onto the immobilized larva. 
	NOTE: Hemolyph-like solutions (HL3 or HL6) may also be used in the place of Schneider&#39;s solution32,33.</li>
	<li>Provide access through the dorsal side of the abdominal body wall of the larva by use of a minutien pin to incise into the midline of the wall.</li>
	<li>Use spring scissors to cut the body wall open from the incision point. Cut along the midline in the anterior-posterior direction34.</li>
	<li>Use fine forceps and spring scissors to disembowel the larva: remove the internal organs including the salivary glands, trachea and gut. Wash the larva twice with Schneider&#39;s insect medium.</li>
	<li>With fine forceps, hold the two body wall flaps, gently stretch them out, and stick two minutien pins on each side. This should produce a hexagon-shaped preparation (Figure 2A).</li>
	<li>Detach the brain from the ventral nerve cord using spring scissors to decrease the possibility of muscle movement during imaging; this also serves to hold the preparation flat against the imaging dish. Use the curved tweezers to push all six minutien pins into the PDMS base. 
	NOTE: Only a small portion of the pins should be embedded within the abdominal wall.</li>
	<li>To confirm that the larva has survived the dissection procedure, slightly poke the ventral nerve cord, this should elicit a peristaltic contraction of the larva abdominal wall.</li>
</ol>
3. Slightly Oblique Illumination Single-Particle Tracking Microscopy
<ol>
	<li>Invert the dissected larva-PDMS base onto a glass-bottomed culture dish. Fill the imaging dish with 2 mL of room temperature Schneider&#39;s insect medium (Figure 2B).</li>
	<li>Locate motor neurons in the second and third segments with synapses on abdominal muscles 6 and 735 using the 63x/1.2 NA water-immersion objective on a fluorescent total internal reflection fluorescence (TIRF) microscope.</li>
	<li>Use the eyepieces of the microscope to locate the dissected larva; identify muscle 6 by use of bright field illumination.</li>
	<li>Use the microscope acquisition software (see Table of Materials), in TIRF configuration; slide the collimator camera angle to 1&#176; to increase the penetration depth, otherwise move out of TIRF critical angle to achieve oblique illumination.</li>
	<li>Switch the 488 nm laser on to visualize mEos2 in green. Use a low laser power (less than 5% of 22.8 mW) to avoid bleaching the green fluorescence. Locate the neuromuscular junctions (which look like beads on a string) and acquire a low resolution TIRF image.</li>
	<li>Simultaneously switch on the 405 nm laser (photoconverts mEos2 from green to red species) and the 561 nm laser (image the photoconverted red mEos2) to stochastically localize single mEos2 fluorescent proteins. 
	NOTE: Use of low 405 nm laser power is recommended (0.005 to 0.9% of 4.78 mW) as this allows for a relatively constant photoconversion rate during movie acquisition. Use between 50 and 75% of the 561 nm laser (20.1 mW), as this is sufficient to acquire fluorescence from the red mEos2 species without adversely affecting the morphology of the muscle tissue surrounding the neuromuscular junction.</li>
	<li>For each neuromuscular junction chain, acquire a time series composed of 15,000 frames or more at 33 Hz. Save as.czi format in order to preserve the accompanying meta-data for possible reference.</li>
	<li>Use ImageJ to convert &#39;.czi&#39; movie files to &#39;.tiff&#39; files.</li>
	<li>Analyze the acquired movies with suitable single-molecule tracking analytical software11,14,36 such as TrackMate on FIJI/ImageJ37 (see Table of Materials). Locates the &#39;x&#39; and &#39;y&#39; coordinates of each localization by the Gaussian fit of the intensity point spread function (PSF). 
	Note: Troubleshooting: If green fluorescence is not observed or is too dim at the neuromuscular junction upon exposure to the 488 nm laser, briefly switch on the photoconverting and imaging lasers (405 nm and 561 nm), as this helps to produce more of the red species of mEos2. Then switch back to the 488 nm laser and take a TIRF image.</li>
</ol>
4. Single-Molecule Localization in Fixed Larvae
<ol>
	<li>To measure the protein nanocluster size via PALM, replace the Schneider&#39;s insect medium after dissection with a fixative - 4% paraformaldehyde diluted in phosphate buffered saline (PBS).</li>
	<li>Incubate the larva for 30 min at room temperature.</li>
	<li>Remove the dissection pins and place the fixed larva in a 1.7 mL clear snap-lock microcentrifuge tube.</li>
	<li>Dissect and fix as many other larvae as needed, then wash them three times with PBS.</li>
	<li>Replace the PBS with block buffer (a solution of PBS, 0.1% Triton X-100, and 3% normal goat serum).</li>
	<li>Wash the larvae with PBS three times, then incubate with the necessary primary antibody (diluted in the block buffer solution).</li>
	<li>Incubate overnight at 4 oC.</li>
	<li>Wash the larvae three times with PBS, then incubate with the necessary secondary antibody (diluted in the block buffer solution).</li>
	<li>Wash the larvae three times with PBS, attach the larva back onto the PDMS base, and image as previously described for single-molecule tracking.</li>
</ol>

The authors have nothing to disclose.

This protocol describes single-molecule tracking of mEos2-tagged proteins at the neuromuscular junction of the Drosophila third instar larva. To avoid over-expression of transgenic protein, Syntaxin-1A-mEos2 expression was driven by endogenous Syntaxin-1A promoter. Studies of synaptic protein mobility have mostly been limited to in vitro investigations of cultured cells and cortical neurons9,11,12,13. Whether these proteins exhibit similar mobility signatures in a live animal is largely unknown. To visualize single-protein mobility in vivo, limitations such as optical transparency, accessibility, and penetration depth have to be overcome. By dissecting the larval preparation and visualizing the neuromuscular junctions embedded on the surface of the muscle, we avoid the issue of optical transparency and accessibility. Attempts to image single-molecule mobility in normal TIRF configuration proved unsuccessful. However, use of slightly oblique illumination allows for increased excitation laser penetrating depth. Furthermore, the minutien pins used to immobilize the larva helped to avoid any possible adverse effect of anesthetic use on protein mobility. It is possible to use higher magnification objectives, such as the 100x oil immersion objectives, to visualize single molecules in vivo. However, increased magnification may increase the occurance of drifts out of focus. In addition, we have used a lower intensity (~30%) 561 nm laser successfully to image single molecules at motor nerve terminals. Additional testing may be required to use lower laser power.
This protocol is suitable to visualize the mobility of synaptic proteins within the proximity of the neuronal plasma membrane. Using this approach, the mobility of the SNARE protein - Syntaxin-1A and the autophagosome bound protein Atg18a - have been successfully imaged in vivo14,26. The mobility of proteins on motor neuron axons can also be investigated27. However, investigation of the mobility of proteins on the brain or ventral nerve cord may prove difficult to assess due to the high background signal produced by the underlying tissue; in addition, visualizing protein mobility on the same brain structure will require fluorescently tagging the brain structure (to ensure replicability), and use of dual-camera imaging would be needed.
Current methods available for super-resolution microscopy in Drosophila are mostly limited to imaging embryos, rather than the more developed third instar larva38,39. The main issue with these protocols is that they yield a low number of trajectories in the area imaged, and hence prevent in-depth analysis of mobility states22,40. Our in vivo single molecule tracking protocol has the capacity to generate a high number of trajectories, and could therefore be used in other animal models such as Caenorhabiditis elegans and zebrafish. Indeed, every NMJ chain generated ~2,400 trajectories making it suitable for analyzing the different mobile states and transitions between these states14,27. Additionally, many in vivo single molecule imaging strategies rely on the use of anesthetics to immobilize the animals20,22, which might alter the physiologic function under which the imaging is done. Here we optimized an &#39;anesthesia-free&#39; protocol to immobilize the larvae using minutien pins.
A potential use of this protocol may be for visualizing the mobility of proteins in three dimensions. Recent advances in super-resolution microscopy allow protein mobility to be visualized in vitro in &#39;x&#39;, &#39;y&#39;, and &#39;z&#39; dimensions41,42. Our current method does not account for the mobility of proteins in the &#39;z&#39; axis. Yet the Drosophila motor nerve terminal is a spherical structure and a valuable future approach will be to quantify protein mobility in a three-dimensional volume43. Imaging in the z-dimension is feasible using an astigmatic lens. Alternatively, in TIRF mode44,45, the z-resolution is also increased. However, in our experiments, we have used slightly oblique illumination to visualize single molecules of syntaxin1A-mEos2 without the astigmatic lens.
In conclusion, availability of both a transgenic Drosophila fly stock expressing proteins tagged with a photoconvertible protein, a PDMS base to dissect the transgenic larva, as well as a TIRF microscope equipped with the possibility of changing the angle of illumination are the most important tools needed to visualize single proteins as described in this protocol.

Neuronal communication relies on neurotransmitter release, which occurs through the regulated fusion of neurotransmitter-containing synaptic vesicles with the plasma membrane1. This process called exocytosis2,3,4,5,6 is highly dynamic and can be up- or down-regulated according to fluctuations in the rate of stimulation7. The proteins involved in these processes are subject to Brownian motion, and therefore display nanoscopic organization capable of sustaining a range of binding actions that underpin these physiological functions. Plasma membrane proteins are highly dynamic, allowing lateral trapping of molecules into nanoclusters on the plasma membrane7,8,9,10,11,12. As such, investigating the mobility of these proteins helps to elucidate their mode of action8. Synaptic proteins such as the soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs), for example Syntaxin-1A, are now known to exhibit lateral fast mobility, as well as lateral trapping within nanoclusters that may serve as molecular depots and sites for vesicle fusion9,13,14,15.
The recent development of photoconvertible fluorescent proteins, such as monomeric (m)Eos16,17 and Kaede18, has allowed the nanoscopic resolution of neuronal plasma membrane proteins through the use of approaches such as photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)14,15,19. These developments have enabled investigations into the mobility and nanoclustering of biological proteins in cultured live cells and neurons. More recently, studies have been carried out to elucidate (at similar high resolutions) the dynamics and organization of these membrane proteins in the neurons of live intact organisms such Mus musculus, Caenorhabditis elegans, and Drosophila melanogaster14,20,21,22.
Investigating the dynamics and organization of a protein in vivo requires not only use of the recently developed super-resolution imaging tools (such as PALM, STORM, and photoconvertible fluorophores), but also the ability to overcome hindrances such as accessibility of the protein and the penetration depth of the imaging laser. Imaging intracellular proteins in an intact animal is inherently challenging as is imaging proteins on structures embedded deep within living tissues of the intact animal, such as the hippocampus of an intact mouse. Hence, proteins on or close to the surface of the tissue are more easily imaged. Another difficulty with imaging in vivo is in ensuring reproducibility across animals. As the anatomy might differ from one sample to the other, selecting a structure with ease of replication in different intact animal samples could also prove challenging. The use of stereotypical structures, such as synapses, help with overcoming some of these difficulties.
Recent studies have imaged protein mobility in animals with an optically transparent epidermis such as Caenorhabditis elegans22, while other investigations have imaged protein organization on the surface of a tissue/organ, for example actin imaging in cortical neurons through the skull of a live mouse21. In some cases, anesthetics have been used to keep the animal immobile; however, specific anesthetics may adversely affect the protein of interest. Alternative means to keep animals immobile during in vivo imaging should therefore be explored to minimize error.
Another caveat to super-resolution imaging in vivo is that the lasers used to illuminate the proteins of interest could adversely affect the surrounding tissue, and hence keeping the excitation intensity as low as possible is recommended. Illumination of the surrounding tissue by the laser also tends to increase the background signal. The use of low excitation intensity helps reduce the background, the caveat being it could also lead to a decrease in fluorescent protein photon yield. Background subtraction during analysis could be used as an alternative means to reduce the background signal prior to image analysis.
Drosophila presents an ideal organism for in vivo imaging as it provides well-established genetic tools for the investigation of specific protein function. The upstream activating sequence (UAS)-Gal4 system enables temporal and spatial expression of proteins and can therefore be used to manipulate neurotransmission23, such that thermo-genetic stimulation using Drosophila transient receptor potential sub-family A1 (dTRPA1)24 or opto-genetic stimulation using far-red-shifted CsChrimson25 can be combined with imaging14. We have overcome many of the complications of performing in vivo single-molecule imaging in this system and now describe how single-particle tracking can be carried out in Drosophila melanogaster third instar larvae.

<table><tbody><tr><td>PDMS (Sylgard 184 silicone elastomer kit)</td><td>Dow Corning Corporation</td><td>000000000001064291</td><td></td></tr><tr><td>Glass-bottomed Culture Dish</td><td>In Vitro Scientific</td><td>D29-20-1.5-N</td><td></td></tr><tr><td>Optical Stereo-microscope</td><td>Olympus</td><td>SZ51</td><td></td></tr><tr><td>Curved Tweezers (Style 5/45)</td><td>Electron Microscopy Sciences</td><td>0208-5AC-PO</td><td></td></tr><tr><td>Minutien Pins (0.1mm)</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Schneider&#039;s Insect Medium</td><td>Sigma, Life Sciences</td><td>S0146</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>Fine Forceps (Dumont #5)</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>ELYRA PS.1 Microscope</td><td>Zeiss</td><td>PS.1</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Lonza</td><td>17-515Q</td><td></td></tr><tr><td>Paraformaldehyde (16%)</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T-9284</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>Maxyclear snaplock microcentrifuge Tube (1.7 mL)</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>Zen Black acquisition software</td><td>Carl Zeiss, 2012</td><td></td><td></td></tr><tr><td>Metamorph software</td><td>Molecular Devices</td><td>7.7.8</td><td>PALM Tracer Plugin</td></tr><tr><td>Fly strain</td><td></td><td>w&lt;sup&gt;1118&lt;/sup&gt;; HA-Sx1A-mEos2</td><td></td></tr></tbody></table>

in vivo single-molecule tracking at the drosophila presynaptic motor nerve terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

Using the protocol listed above, Syntaxin-1A-mEos2 transgenic Drosophilamelanogaster were generated (Figure 1). Transgenic third instar Drosophila larvae were dissected on the PDMS cylindrical base (Figure 2A-F). To visualize single molecules of Syntaxin-1A, we used PALM on a TIRF microscope with slightly oblique laser illumination14.
Single molecules of Syntaxin-1A were tracked on the presynaptic motor nerve terminals on muscle 6 of the second abdominal segment. The green fluorescence of mEos2 could be detected as shown by the low-resolution image of Syntaxin-1A-mEos2 on type 1b boutons when excited using the 488 nm laser (Figure 3A). In the imaging experiments, the 405 nm and 561 nm excitation laser imaging depths were 133.5 nm and 165.6 nm, respectively. The green image was acquired as an average of 16 individual frames. This green image was used to create the region of interest used to limit analysis of localizations in the photoconverted movie to the presynaptic motor nerve terminals alone.
The analysis of the acquired photoconverted sptPALM movies generated a super-resolved intensity, diffusion coefficient, and trajectory maps (Figure 3A). Syntaxin-1A-mEos2 mobility was further quantified by analysis of the mean square displacement (MSD) of the individual trajectories (Figure 3B). Statistical comparisons can be done using the area under the MSD curve (Figure 3B). Further analysis of mobility yields the diffusion coefficient distribution of Syntaxin-1A-mEos2, and this can be statistically evaluated by comparison of the mobile to the immobile populations (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56952/56952fig1.jpg" /> 
Figure 1: Generating mEos2-tagged constructs. The pFL44S-attB-MCS-w+ vector is linearized with BamHI and XbaI. Overlapping polymerase chain reaction (PCR) fragments encoding the protein of interest (POI) and mEos2, respectively, can be cloned, for example, by in vivo recombination in ura3- yeast cells or by Gibson assembly. Note that to generate the Syntaxin-1A-mEos2 transgene, we cloned the construct flanked by the endogenous Syntaxin-1A promoter and terminator sequences. The resulting construct can subsequently be injected into Drosophila embryos to create a transgenic fly line expressing the protein of interest (POI) fused to mEos2. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56952/56952fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56952/56952fig2.jpg" /> 
Figure 2: Drosophila larva dissection and oblique illumination. (A) Schema showing Drosophila larva dissection carried out on half-cylindrical PDMS base. A filleted larva was held onto the PDMS gel by use of minutien pins. (B-C) The larva-PDMS prep was placed inside a glass-bottomed culture dish containing Schneider&#39;s insect medium. A TIRF microscope in a slightly oblique illumination configuration was used to visualize Syntaxin-1A-mEos2 in the motor nerve terminals. (D-F) A half-cylindrical PDMS base with dissected Drosophila larva. The larva-PDMS preparation was placed in an imaging dish filled with Schneider&#39;s medium, and the larva was imaged on the super-resolution PALM microscope. Scale bar, 10 mm. (This figure has been modified from14). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56952/56952fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56952/56952fig3.jpg" /> 
Figure 3: In vivo tracking of Syntaxin-1A-mEos2 at the presynaptic motor nerve terminal. (A) A low-resolution TIRF image of Syntaxin-1A-mEos2 on a type 1b motor nerve terminal. Analysis of photoconversion movies imaged at 33 Hz generated sptPALM super-resolved intensity, diffusion coefficient, and trajectory maps. 5,309 individual trajectories were imaged over 16,000 frames movie acquisition. Scale bar, 3 &#956;m. (B-C) Mean square displacement (MSD) of Syntaxin-1A-mEos2 was plotted from 6 different motor nerve terminals; area under the MSD curve (AUC) was used to quantify the level of mobility. The diffusion coefficient distribution reveals mobile and immobile Syntaxin-1A populations that can be quantified as ratios of each other; an average of ~2,400 trajectories were obtained per motor nerve terminal (n = 3 larvae). Data presented as mean standard error of mean. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56952/56952fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal at JoVE.com

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

in-vivo-single-molecule-tracking-at-drosophila-presynaptic-motor

Research

JoVE Journal

Neuroscience

8.4K Views.  The University of Queensland. Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

Video: In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

Developmental Biology

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

Biology

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

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

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

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

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

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

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 ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

Viewing Microtubule Networks in Drosophila Neuromuscular Junctions and Muscle Cells

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

Author Spotlight: Understanding Microtubule Network in Drosophila Neuromuscular Junctions 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with ...

Microelectrode-Mediated FM Dye Loading and Unloading in Drosophila Larvae Nerve Cells

Source: Kopke, D. L., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57765">FM dye cycling at the synapse: comparing high potassium depolarization, electrical and channelrhodopsin stimulation.</a> J. Vis. Exp. (2018)
In this video, Drosophila larvae nerve fibers are immersed in FM dye, which binds to cell membrane lipids. Suction microelectrode and electrical pulses stimulate the endocytosis process, incorporating the FM dye into newly formed endocytic vesicles. This method, which visualizes presynaptic stages of neurotransmission, is useful for studying synaptic function and neurotransmitter release.

1. Larval Glue Dissection

	Thoroughly mix 10 parts of silicone elastomer base with 1 part of silicone elastomer curing agent from the elastomer kit.
	...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

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In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

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

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

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

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

Microelectrode-Mediated FM Dye Loading and Unloading in Drosophila Larvae Nerve Cells