Name: FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation (Video) | JoVE Journal
Uploaded: 2018-05-24T15:00:00.000+00:00
Duration: 8 min 31 s
Description: Learn FM dye cycling at the Drosophila larval neuromuscular junction using three stimulation methods: high potassium depolarization, electrical stimulation, and optogenetics. This technique enables optical tracking of synaptic vesicle endo- and exocytosis in live preparations.

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We thank Broadie Lab members for contributions to this article. This work was supported by NIH R01s MH096832 and MH084989 to K.B., and NIH predoctoral fellowship F31 MH111144 to D.L.K.

1. Larval Glue Dissection
<ol>
	<li>Thoroughly mix 10 parts of silicone elastomer base with 1 part of silicone elastomer curing agent from the elastomer kit (Table of Materials).</li>
	<li>Coat 22 x 22 mm glass coverslips with the elastomer and cure on a hot plate at 75 &#730;C for several hours (until no longer sticky to the touch).</li>
	<li>Place a single elastomer-coated glass coverslip into the custom-made plexi glass dissection chamber (Figure 1, bottom) in preparation for the larval dissection.</li>
	<li>Prepare the glue pipettes from borosilicate glass capillary using a standard microelectrode puller to obtain the desired taper and tip size.</li>
	<li>Gently break off the micropipette tip, and to the other end, attach 2 ft of flexible plastic tube (1/32&#34; interior diameter, ID; 3/32&#34; outside diameter, OD; 1/32&#34; wall; Table of Materials) with mouth fitting (P2 pipette tip).</li>
	<li>Fill a small container (0.6 mL Eppendorf tube cap) with a small volume (~20 &#181;L) of glue (Table of Materials) in preparation for the larval dissection.</li>
	<li>Fill the chamber with saline (in mM): 128 NaCl, 2 KCl, 4 MgCl2, 1 CaCl2, 70 sucrose, and 5 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) pH 7.2.</li>
	<li>Add anti-horse radish peroxidase (HRP) antibody conjugated to Alexa Fluor 647 (anti-HRP:647; dilute 1:10 from a 1 mg/mL stock) for labeling the NMJ presynaptic terminal during dissection21,22.</li>
	<li>Using a fine paintbrush (size 2), remove a wandering third instar from the food vial and place onto the elastomer-coated cover glass.</li>
	<li>Fill the glass micropipette tip with a small volume of glue using negative air pressure generated by mouth with attachment (step 1.5).</li>
	<li>Position larva dorsal side up with forceps and glue the head to the elastomer-coated coverslip with a small drop of glue using positive air pressure by mouth.</li>
	<li>Repeat this procedure with the posterior end of the larva, making sure that the animal is stretched taut between the two glue attachments.</li>
	<li>Using scissors (blades 3 mm; Table of Materials), make a horizontal cut (~1 mm) at posterior and a vertical cut all along the dorsal midline.</li>
	<li>Using fine forceps (#5, Table of Materials), gently remove dorsal trachea, gut, fat body and other internal organs covering the musculature.</li>
	<li>Repeat the gluing procedure for the four body wall flaps, making sure to gently stretch the body wall both horizontally and vertically.</li>
	<li>Lift the ventral nerve cord (VNC) using forceps, carefully cut the motor nerves with the scissors, and then completely remove the VNC.</li>
	<li>Replace the dissection saline with Ca2+-free saline (same as the above dissection saline without the CaCl2) to stop SV cycling.</li>
</ol>
2. Option 1: High [K+] FM Dye Loading
<ol>
	<li>From a FM1-43 stock solution (4 mM), add 1 &#181;L to 1 mL of 90 mM KCl solution (high [K+] in dissection saline) for a final concentration of 4&#160;&#181;M.</li>
	<li>Using a pipette, replace the Ca2+-free saline in the imaging chamber with the high [K+] FM dye solution to stimulate SV endocytosis dye uptake.</li>
	<li>Immediately start a digital timer for the pre-determined duration of the high [K+] depolarizing stimulation period (e.g., 5 min; Figure 2).</li>
	<li>To confirm a healthy larval preparation, note the strong contractions of the musculature for the duration of the high [K+] depolarization period.</li>
	<li>When the timer period ends, quickly remove the high [K+] FM dye solution and replace with Ca2+-free saline to stop SV cycling.</li>
	<li>Wash in quick succession with the Ca2+-free saline (5x for 1 min) to ensure the high [K+] FM dye solution is completely removed.</li>
	<li>Maintain the larval preparation in fresh Ca2+-free saline for immediate imaging with the confocal microscope.</li>
</ol>
3. Imaging: Confocal Microscopy
<ol>
	<li>Use an upright confocal microscope with a 40X water immersion objective to image NMJ dye fluorescence (other microscopes can be used).</li>
	<li>Image muscle 4 NMJ of abdominal segments 2-4 (other NMJs can be imaged) and collect images using appropriate software (Table of Materials).</li>
	<li>Use a HeNe 633 nm laser to excite HRP:647 (with long-pass filter &#62; 635 nm) and an Argon 488 nm laser to excite FM1-43 (with bandpass filter 530-600&#160;nm).</li>
	<li>Operationally determine optimal gain and offset for both channels. 
	NOTE: These settings will remain constant throughout the rest of the experiment.</li>
	<li>Take a confocal Z-stack through the entire selected NMJ from the HRP-marked top to bottom of the synaptic terminal.</li>
	<li>Take careful note of the NMJ imaged (segment, side and muscle) to ensure excess to the exact same NMJ after FM dye unloading.</li>
</ol>
4. High [K+] Stimulation: FM Dye Unloading
<ol>
	<li>Replace Ca2+-free saline with the high [K+] saline (without FM1-43 dye) to drive depolarization, SV exocytosis and dye release.</li>
	<li>Immediately start a digital timer for the pre-determined duration of the high [K+] stimulation period (e.g., 2 min; Figure 2).</li>
	<li>When the timer period ends, immediately remove the high [K+] saline and replace with Ca2+-free saline to stop SV cycling.</li>
	<li>Wash in quick succession with Ca2+-free saline (5x for 1 min) to ensure the high [K+] saline is completely removed.</li>
	<li>Maintain the larval preparation in fresh Ca2+-free saline for immediate imaging with the confocal microscope.</li>
	<li>Be certain to image the FM1-43 dye fluorescence at the same NMJ noted above using the same confocal settings.</li>
</ol>
5. Option 2: Electrical Stimulation FM Dye Loading
<ol>
	<li>Prepare a suction pipette using the microelectrode puller (Table of Materials) to obtain the required taper and tip size.</li>
	<li>Fire-polish the microelectrode tip with a micro-forge until a single motor nerve can be sucked up with a tight fit.</li>
	<li>Slide suction pipette onto the electrode holder on a micromanipulator and attach to the long flexible plastic tube and a syringe.</li>
	<li>Set stimulator parameters (e.g., 15 V, 20 Hz frequency, 20 ms duration and time of 5 min (Figure 2) or 1 min (Figure 3)).</li>
	<li>Replace the Ca2+-free saline on the larval preparation with above FM1-43 saline (4 &#181;M; 1 mM CaCl2) on the electrophysiology rig.</li>
	<li>Put the preparation on the microscope stage and raise the stage until the larva and suction pipette are in focus (40X water-immersion objective).</li>
	<li>Suck up a loop of cut motor nerve innervating the selected hemisegment with negative air pressure generated by the syringe into the suction electrode.</li>
	<li>Test the suction electrode function with a short burst of stimulation while visually monitoring for the muscle contraction in the selected hemisegment.</li>
	<li>Stimulate the motor nerve using selected parameters (step 5.4) to drive SV endocytosis and FM1-43 dye uptake (Figure 2).</li>
	<li>Wash in quick succession with Ca2+-free saline (5x for 1 min) to ensure the FM1-43 dye solution is completely removed.</li>
	<li>Maintain the larval preparation in fresh Ca2+-free saline for immediate imaging using the confocal imaging protocol from above.</li>
	<li>Take careful note of the NMJ imaged (segment, side and muscle) to ensure access to the exact same NMJ after FM dye unloading.</li>
</ol>
6. Electrical Stimulation: FM Dye Unloading
<ol>
	<li>Replace the Ca2+-free saline with regular saline (without FM1-43 dye)and place the preparation back on the electrophysiology rig stage.</li>
	<li>Set the stimulator parameters for unloading (e.g., 15 V, 20 Hz frequency, 20 ms duration and time of 2 min (Figure 2) or 20 s (Figure 3)).</li>
	<li>Suck the same motor nerve into the same electrode as above, and then stimulate to activate SV exocytosis and FM1-43 dye release.</li>
	<li>Wash in quick succession with Ca2+-free saline (5x for 1 min) to ensure the external dye is completely removed.</li>
	<li>Maintain the larval preparation in fresh Ca2+-free saline for immediate imaging with the confocal microscope.</li>
	<li>Ensure to image the FM1-43 dye fluorescence at the same NMJ noted above using the same confocal settings.</li>
</ol>
7. Option 3: Channelrhodopsin Stimulation FM Dye Loading
<ol>
	<li>Raise ChR2-expressing larvae on food containing the ChR2 co-factor all-trans retinal (dissolved in ethanol; 100 &#181;M final concentration).</li>
	<li>Place the larval preparation in the plexiglass chamber on a dissection microscope stage equipped with a camera port.</li>
	<li>Attach a blue LED (470 nm; Table of Materials) to a programmable stimulator using a coaxial cable and place the LED into the camera port.</li>
	<li>Focus the blue LED light beam onto the dissected larval function using the microscope zoom function.</li>
	<li>Replace the Ca2+-free saline on the larval preparation with above FM1-43 saline (4 &#181;M; 1 mM CaCl2) on the optogenetic stage.</li>
	<li>Set the LED parameters using the stimulator (e.g., 15 V, 20 Hz frequency, 20 ms duration and time of 5 min (Figure 2)).</li>
	<li>Start the light stimulation and track with a timer for the pre-determined duration of the optogenetic stimulation period (e.g., 5 min; Figure 2).</li>
	<li>When the timer stops, quickly remove the FM dye solution and replace with Ca2+-free saline to stop the SV cycling.</li>
	<li>Wash in quick succession with the Ca2+-free saline (5x for 1 min) to ensure the FM dye solution is completely removed.</li>
	<li>Maintain the larval preparation in fresh Ca2+-free saline for immediate imaging with the confocal microscope using imaging protocol from above.</li>
	<li>Take careful note of the NMJ imaged (segment, side and muscle) to ensure access to the exact same NMJ after FM dye unloading.</li>
</ol>
8. Channelrhodopsin Stimulation: FM Dye Unloading
<ol>
	<li>Replace Ca2+-free saline with regular saline (without FM1-43 dye)on the dissection microscope stage with camera port LED focused on the larva.</li>
	<li>Set the stimulator parameters for unloading (e.g., 15 V, 20 Hz frequency, 20 ms duration and time of 2 min (Figure 2)).</li>
	<li>Start the light stimulation and track with a timer for the pre-determined duration of the optogenetic stimulation period (e.g., 2 min; Figure 2).</li>
	<li>When the timer period ends, quickly remove the FM dye solution and replace with Ca2+-free saline to stop the SV cycling.</li>
	<li>Wash in quick succession with Ca2+-free saline (5x for 1 min) to ensure the external dye is completely removed.</li>
	<li>Maintain the larval preparation in fresh Ca2+-free saline for immediate imaging with the confocal microscope.</li>
	<li>Ensure to image the FM1-43 dye fluorescence at the same NMJ noted above using the same confocal settings.</li>
</ol>
9. Fluorescence Quantification
<ol>
	<li>Load the image in Image J (NIH open source) and create a maximum intensity projection by clicking Image | Stacks | Z Project.</li>
	<li>Using the anti-HRP:647 channel, go to Image | Adjust | Threshold and slide the top tool bar until just the NMJ is highlighted.</li>
	<li>Using the wand tool, click on the NMJ. If the NMJ is discontinuous, hold the Shift button and select all parts.</li>
	<li>Change the image to the FM1-43 dye channel and go to Analyze | Measure to obtain the fluorescence measurement.</li>
	<li>Repeat steps 9.1-9.4 for the &#34;unloaded&#34; image from the same NMJ (identified segment, side and muscle).</li>
	<li>To obtain the percentage of dye that was unloaded, take the ratio of the unloaded/loaded fluorescence intensities. 
	NOTE: This procedure can be modified to analyze fluorescence on a bouton-per-bouton basis using either the &#34;oval&#34; or &#34;freehand&#34; selection tools. Background fluorescence can be subtracted by sampling the muscle fluorescence. Agents can also be added to reduce this background.</li>
</ol>

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<li>Baldwin, M. L., Rostas, J. A. P., Sim, A. T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+modes+of+exocytosis+from+synaptosomes+are+differentially+regulated+by+protein+phosphatase+types+2A+and+2B%5BTitle%5D)+AND+%22J+Neurochem%22%5BJournal%5D)">Two modes of exocytosis from synaptosomes are differentially regulated by protein phosphatase types 2A and 2B.</a> J Neurochem. 85 (5), 1190-1199 (2003).</li>
<li>Nagel, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Channelrhodopsin-2%2C+a+directly+light-gated+cation-selective+membrane+channel%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.</a> Proc Natl Acad Sci U S A. 100 (24), 13940-13945 (2003).</li>
<li>Pfeiffer, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tools+for+neuroanatomy+and+neurogenetics+in+Drosophila%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Tools for neuroanatomy and neurogenetics in Drosophila.</a> Proc Natl Acad Sci U S A. 105 (28), 9715-9720 (2008).</li>
<li>Nagel, G., Brauner, M., Liewald, J. F., Adeishvili, N., Bamberg, E., Gottschalk, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Light+Activation+of+Channelrhodopsin-2+in+Excitable+Cells+of+Caenorhabditis+elegans+Triggers+Rapid+Behavioral+Responses%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Light Activation of Channelrhodopsin-2 in Excitable Cells of Caenorhabditis elegans Triggers Rapid Behavioral Responses.</a> Curr Biol. 15 (24), 2279-2284 (2005).</li>
<li>Lin, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+user%27s+guide+to+channelrhodopsin+variants%3A+features%2C+limitations+and+future+developments%5BTitle%5D)+AND+%22Exp+Physiol%22%5BJournal%5D)">A user's guide to channelrhodopsin variants: features, limitations and future developments.</a> Exp Physiol. 96 (1), 19-25 (2011).</li>
<li>Sullivan, K. M., Scott, K., Zuker, C. S., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ryanodine+receptor+is+essential+for+larval+development+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">The ryanodine receptor is essential for larval development in Drosophila melanogaster.</a> Proc Natl Acad Sci U S A. 97 (11), 5942-5947 (2000).</li>
<li>Narahashi, T., Moore, J. W., Scott, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tetrodotoxin+Blockage+of+Sodium+Conductance+Increase+in+Lobster+Giant+Axons%5BTitle%5D)+AND+%22J+Gen+Physiol%22%5BJournal%5D)">Tetrodotoxin Blockage of Sodium Conductance Increase in Lobster Giant Axons.</a> J Gen Physiol. 47 (5), 965-974 (1964).</li>
<li>Narahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemicals+as+tools+in+the+study+of+excitable+membranes%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Chemicals as tools in the study of excitable membranes.</a> Physiol Rev. 54 (4), 813-889 (1974).</li>
<li>Kuromi, H., Yoshihara, M., Kidokoro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+inhibitory+role+of+calcineurin+in+endocytosis+of+synaptic+vesicles+at+nerve+terminals+of+Drosophila+larvae%5BTitle%5D)+AND+%22Neurosci+Res%22%5BJournal%5D)">An inhibitory role of calcineurin in endocytosis of synaptic vesicles at nerve terminals of Drosophila larvae.</a> Neurosci Res. 27 (2), 101-113 (1997).</li>
<li>Seamon, K. B., Daly, J. W. <a target="_blank" href="http://www.cell.com/trends/pharmacological-sciences/abstract/0165-6147(83)90326-7">Forskolin, cyclic AMP and cellular physiology.</a> Trends Pharmacol Sci. 4, 120-123 (1983).</li>
<li>Zhang, D., Kuromi, H., Kidokoro, Y. <a target="_blank" href="https://link.springer.com/chapter/10.1007/978-4-431-66973-9_32">Synaptic Transmission at the Drosophila Neuromuscular Junction: Effects of Metabotropic Glutamate Receptor Activation.</a> Slow Synaptic Responses Modul. , 260-265 (2000).</li>
<li>Kuromi, H., Kidokoro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+optically+determined+size+of+exo%2Fendo+cycling+vesicle+pool+correlates+with+the+quantal+content+at+the+neuromuscular+junction+of+Drosophila+larvae%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.</a> J Neurosci. 19 (5), 1557-1565 (1999).</li>
<li>Kuromi, H., Kidokoro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+Replenishment+of+Two+Vesicle+Pools+Depends+on+the+Source+of+Ca2%2B+at+the+Drosophila+Synapse%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Selective Replenishment of Two Vesicle Pools Depends on the Source of Ca2+ at the Drosophila Synapse.</a> Neuron. 35 (2), 333-343 (2002).</li>
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The authors declare no competing interests.

High [K+] saline depolarizing stimulation is by far the easiest of the three options for activity-dependent FM dye cycling, but likely the least physiological29. This simple method depolarizes every accessible cell in the entire animal, and so does not allow directed studies. It may be possible to locally apply high [K+] saline with a micropipette, but this will still depolarize pre/postsynaptic cells and likely synapse-associated glia1. Another major ...

The beautifully-characterized Drosophila larval neuromuscular junction (NMJ) glutamatergic synapse model has been used to study synapse formation and function with a vast spectrum of genetic perturbations1. The motor neuron terminal consists of multiple axon branches, each with many enlarged synaptic boutons. These capacious varicosities (up to 5 &#181;m in diameter) contain all of the neurotransmission machinery, including uniform glutamatergic synaptic vesicles (SVs; ~40 nm in diameter) in cytosolic reserve and readily-releasable pools2. These vesicles dock at the presynaptic plasma membrane fusion site active zones (AZs), where exocytosis mediates the glutamate neurotransmitter release for trans-synaptic communication. Subsequently, the SVs are retrieved from the plasma membrane via kiss-and-run recycling or clathrin-mediated endocytosis (CME) for repeated exo/endocytosis cycles. The Drosophila NMJ is easily accessible and well-suited for both isolating and characterizing SV cycle mutants. Using forward genetic screens, novel mutations have led to the identification of new genes critical for the SV cycle3. Moreover, reverse genetic approaches starting with already known genes have led to the elucidation of new SV cycle mechanisms through the careful description of mutant cycling phenotypes4. The Drosophila NMJ is nearly ideal as an experimental synaptic preparation for dissecting SV endocytosis and exocytosis mechanisms via methods to optically track vesicle cycling during neurotransmission.
A range of fluorescent markers allow visual tracking of vesicles during cycling dynamics, but the most versatile are FM dye analogs which is first synthesized by Mao, F., et al.5. Structurally, FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring, with a central region conferring spectral properties. These styryl dyes partition reversibly in membranes, do not &#39;flip-flop&#39; between membrane leaflets and so are never free in the cytosol, and are far more fluorescent in membranes than water5. Reversible insertion into a lipid bilayer causes a 40-fold increase in fluorescence6. At neuronal synapses, classic FM dye labeling experiments consist of bathing the synaptic preparation with the dye during depolarizing stimulation to load dye via SV endocytosis. External dye is then washed away and the SV cycle is arrested in a calcium-free ringer solution to image loaded synapses7. A second round of stimulation in a dye-free bath triggers FM release through exocytosis, a process that can be followed by measuring the fluorescence intensity decrease. SV populations from a single vesicle to pools containing hundreds of vesicles can be quantitatively monitored6,7. FM dyes have been used to dissect activity-dependent mobilization of functionally distinct SV pools, and to compare kiss-and-run vs. CME cycling8,9. The method has been modified to separately assay evoked, spontaneous and miniature synaptic cycle activities (with highly sensitive equipment to detect very small fluorescence changes and reduce photobleaching)10. Assays can be extended to the ultrastructural level by photoconverting the fluorescent FM signal into an electron-dense label for transmission electron microscopy11,12,13,14.
Historically, bathing synaptic preparations in a high concentration of potassium (hereafter referred to as &#34;high [K+]&#34;) has been the method of choice for depolarizing stimulation to induce SV cycling; ranging from the frog cholinergic NMJ5, to cultured rodent brain hippocampal neurons15, to the Drosophila glutamatergic NMJ model16,17. This high [K+] approach is simple, requires no specialized equipment, and is therefore accessible to most labs, but has limitations for both application and data interpretation. A much more physiologically appropriate method is to use suction electrode electrical stimulation of the nerve4,5,12. This approach drives action potential propagation for direct stimulation of the presynaptic nerve terminal, and results can be directly compared to electrophysiological assays of neurotransmission function13,14,15, but requires specialized equipment and is technically much more challenging. With the advent of optogenetics, the use of channelrhodopsin neuronal stimulation has additional advantages, including tight spatiotemporal control of channel expression using the binary Gal4/UAS system20. This approach is technically much easier than suction electrode stimulation and requires nothing more than a very cheap LED light source. Here, we employ imaging of FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide) to both compare and contrast these three different stimulation methods at the Drosophila NMJ: simple high [K+], challenging electrical and new channelrhodopsin approaches.

<table><tbody><tr><td>SylGard 184 Silicone Elastomer Kit</td><td>Fisher Scientific</td><td>NC9644388</td><td>To put on cover glass for dissections</td></tr><tr><td>Microscope Cover Glass 22x22-1</td><td>Fisherbrand</td><td>12-542-B</td><td>To put SylGard on for dissections</td></tr><tr><td>Aluminum Top Hot Plate Type 2200</td><td>Thermolyne</td><td>HPA2235M</td><td>To cure the SylGard</td></tr><tr><td>Plexi glass dissection chamber</td><td>N/A</td><td>N/A</td><td>Handmade</td></tr><tr><td>Borosilicate Glass Capillaries</td><td>WPI</td><td>1B100F-4</td><td>To make suction and glue micropipettes</td></tr><tr><td>Laser-Based Micropipette Puller</td><td>Sutter Instrument</td><td>P-2000</td><td>To make suction and glue micropipettes</td></tr><tr><td>Tygon E-3603 Laboratory Tubing</td><td>Component Supply Co.</td><td>TET-031A</td><td>For mouth and suction pipette</td></tr><tr><td>P2 pipette tip</td><td>USA Scientific</td><td>1111-3700</td><td>For mouth pipette</td></tr><tr><td>0.6-mL Eppendorf tube cap</td><td>Fisher Scientific</td><td>05-408-120</td><td>Used to put glue in for dissection</td></tr><tr><td>Vetbond 3M</td><td>WPI</td><td>vetbond</td><td>Glue used for dissections</td></tr><tr><td>Potassium Chloride</td><td>Fisher Scientific</td><td>P-217</td><td>Forsaline</td></tr><tr><td>Sodium Chloride</td><td>Millipore Sigma</td><td>S5886</td><td>For saline</td></tr><tr><td>Magnesium Chloride</td><td>Fisher Scientific</td><td>M35-500</td><td>For saline</td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Millipore Sigma</td><td>C7902</td><td>For saline</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-3</td><td>For saline</td></tr><tr><td>HEPES</td><td>Millipore Sigma</td><td>H3375</td><td>For saline</td></tr><tr><td>HRP:Alexa Fluor 647</td><td>Jackson ImmunoResearch</td><td>123-605-021</td><td>To label neuronal membranes</td></tr><tr><td>Paintbrush</td><td>Winsor &amp;amp; Newton</td><td>94376864793</td><td>To manipulate the larvae</td></tr><tr><td>Dumont Dumostar Tweezers #5</td><td>WPI</td><td>500233</td><td>Used during dissection</td></tr><tr><td>7 cm McPherson-Vannas Microscissors (blades 3 mm)</td><td>WPI</td><td>14177</td><td>Used during dissection&amp;nbsp;</td></tr><tr><td>FM1-43</td><td>Fisher Scientific</td><td>T35356</td><td>Fluorescent styryl dye</td></tr><tr><td>Digital Timer</td><td>VWR</td><td>62344-641</td><td>For timing FM dye load/unload&amp;nbsp;</td></tr><tr><td>LSM 510 META laser-scanning confocal microscope</td><td>Zeiss</td><td></td><td>For imaging the fluorescent markers</td></tr><tr><td>Zen 2009 SP2 version 6.0</td><td>Zeiss</td><td></td><td>Software for imaging on confocal</td></tr><tr><td>HeNe 633nm laser</td><td>Lasos</td><td></td><td>To excite HRP:647 during imaging</td></tr><tr><td>Argon 488nm laser</td><td>Lasos</td><td></td><td>To excite the FM dye during imaging</td></tr><tr><td>Micro-Forge</td><td>WPI</td><td>MF200</td><td>To fire polish glass micropipettes</td></tr><tr><td>20mL Syringe Slip Tip</td><td>BD</td><td>301625</td><td>To suck up the axon for electrical stimulation.</td></tr><tr><td>Micro Manipulator (magnetic base)</td><td>Narishige</td><td>MMN-9</td><td>To control the suction electrode for electrical stimulation</td></tr><tr><td>Stimulator</td><td>Grass</td><td>S48</td><td>To control the LED and electrical stimulation</td></tr><tr><td>Zeiss Axioskop Microscope</td><td>Zeiss</td><td></td><td>Used during electrical stimulation.</td></tr><tr><td>40X Achroplan Water Immersion Objective</td><td>Zeiss</td><td></td><td>Used during electrical stimulation and confocal imaging</td></tr><tr><td>All-trans Retinal</td><td>Millipore Sigma</td><td>R2500</td><td>Essential co-factor for ChR2</td></tr><tr><td>Zeiss Stemi Microscope with camera port</td><td>Zeiss</td><td>2000-C</td><td>Used during channelrhodopsin stimulation</td></tr><tr><td>LED 470nm</td><td>ThorLabs</td><td>M470L2</td><td>Used for ChR activation</td></tr><tr><td>T-Cube LED Driver</td><td>ThorLabs</td><td>LEDD1B</td><td>To control the LED</td></tr><tr><td>LED Power Supply</td><td>Cincon Electronics Co.</td><td>TR15RA150</td><td>To power the LED</td></tr><tr><td>Optical Power and Energy Meter</td><td>ThorLabs</td><td>PM100D</td><td>To measure LED intensity</td></tr></tbody></table>

fm dye cycling at the synapse: comparing high potassium depolarization, electrical and channelrhodopsin stimulation

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them water-soluble with the ability to reversibly enter and exit membrane lipid bilayers. These styryl dyes are relatively non-fluorescent in aqueous medium, but insertion into the outer leaflet of the plasma membrane causes a &#62;40X increase in fluorescence. In neuronal synapses, FM dyes are internalized during SV endocytosis, trafficked both within and between SV pools, and released with SV exocytosis, providing a powerful tool to visualize presynaptic stages of neurotransmission. A primary genetic model of glutamatergic synapse development and function is the Drosophila neuromuscular junction (NMJ), where FM dye imaging has been used extensively to quantify SV dynamics in a wide range of mutant conditions. The NMJ synaptic terminal is easily accessible, with a beautiful array of large synaptic boutons ideal for imaging applications. Here, we compare and contrast the three ways to stimulate the Drosophila NMJ to drive activity-dependent FM1-43 dye uptake/release: 1) bath application of high [K+] to depolarize neuromuscular tissues, 2) suction electrode motor nerve stimulation to depolarize the presynaptic nerve terminal, and 3) targeted transgenic expression of channelrhodopsin variants for light-stimulated, spatial control of depolarization. Each of these methods has benefits and disadvantages for the study of genetic mutation effects on the SV cycle at the Drosophila NMJ. We will discuss these advantages and disadvantages to assist the selection of the stimulation approach, together with the methodologies specific to each strategy. In addition to fluorescent imaging, FM dyes can be photoconverted to electron-dense signals visualized using transmission electron microscopy (TEM) to study SV cycle mechanisms at an ultrastructural level. We provide the comparisons of confocal and electron microscopy imaging from the different methods of Drosophila NMJ stimulation, to help guide the selection of future experimental paradigms.

Figure 1 shows the work-flow for the activity-dependent FM dye imaging protocol. The experiment always begins with the same larval glue dissection, regardless of the stimulation method subsequently used. Figure 1a is a schematic of a dissected larva, showing the ventral nerve cord (VNC), radiating nerves and repeated hemisegmental muscle pattern. The VNC is removed and the preparation bathed in a 4 µM solution of FM1-43 (<strong ...

Synaptic vesicle (SV) cycling is the core mechanism of intercellular communication at neuronal synapses. FM dye uptake and release are the primary means of quantitatively assaying SV endo- and exocytosis. Here, we compare all the stimulation methods to drive FM1-43 cycling at the Drosophila neuromuscular junction (NMJ) model synapse.

FM dyes are used to study the synaptic vesicle (SV) cycle. These amphipathic probes have a hydrophilic head and hydrophobic tail, making them ...

fm-dye-cycling-at-synapse-comparing-high-potassium-depolarization

Research

JoVE Journal

Neuroscience

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

A new means to measure neurotransmission optically using fluorescent dopamine analogs.

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

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.

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

This video demonstrates a protocol to study synaptic vesicle recycling at neuromuscular junctions in channelrhodopsin-expressing Drosophila larvae using a fluorescent dye. Blue light activates the channelrhodopsins, causing calcium influx and synaptic vesicle exocytosis. The dye integrates into the neuronal membranes and is internalized during endocytosis, resulting in enhanced fluorescence. Further blue light stimulation induces exocytosis and dye release, which is shown by reduced...

Analyzing Synaptic Vesicle Recycling and Fluorescent Dye Uptake in Drosophila Larvae

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.<br...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

Functional site-directed fluorometry is a method to study protein domain motions in real time. Modification of this technique for its application in native cells now allows the detection and tracking of single voltage-sensor motions from voltage-gated Ca2+ channels in murine isolated skeletal muscle fibers.

Functional Site-Directed Fluorometry in Native Cells to Study Skeletal Muscle Excitability

The zebrafish larval neuromuscular junction is an attractive model for studying synaptic physiology. It is amenable to many experimental techniques, including electrophysiology and optical imaging. Here, we describe a protocol for imaging synaptic transmission using a pHluorin-based probe under an upright epifluorescence microscope.

Live Imaging of Synaptic Vesicle Recycling in the Neuromuscular Junction of Dissected Larval Zebrafish

Learn FM dye cycling at the Drosophila larval neuromuscular junction using three stimulation methods: high potassium depolarization, electrical stimulation, and optogenetics. This technique enables optical tracking of synaptic vesicle endo- and exocytosis in live preparations.

FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation

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