This work was supported by the National Institutes of Health: RO1-GM108970 (SLG) and F31-NS087837 (CW).

Statement of research ethics: All experiments involving animals detailed herein are subject to the rules and regulations of the UNC Committee on Animal Care and to NIH standards for the care and use of laboratory animals.
1. Preparation and Plating of Dissociated Cortical Neurons
<ol>
	<li>Euthanize timed-pregnant females by CO2 inhalation followed by cervical dislocation. Remove whole brains from the skulls of embryonic day 15.5 (E15.5) mice and microdissect cortices from each hemisphere. For detailed instructions regarding the microdissection of embryonic mouse cortex please see Viesselmann et al8.</li>
	<li>Place no more than 4 cortices in 1 ml&#160;of dissecting media in a sterile 1.6 ml microtube. Add 120 &#956;l of 10x trypsin and invert the tube 3 - 5 times to mix.</li>
	<li>Using a hemocytometer, count cells to seed cell culture dishes. Note: 1 cortical hemisphere provides approximately three million cells.
	<ol>
		<li>For the SDS-resistant SNARE complex biochemistry assay, plate six million cells per 35 mm dish and utilize two dishes per condition. Note: This is simplified by using 6 well plates when comparing multiple conditions.</li>
		<li>For branching assays, plate neurons on nitric acid washed circular coverslips at a density of 250,000 cells per 35 mm dish, for DIC imaging of axon branching plate at a density of 150,000. These densities avoid overcrowding and simplify analysis.</li>
	</ol>
	</li>
	<li>For live cell TIRF microscopy, resuspend two million neurons per transfection in nucleofection solution at RT.
	<ol>
		<li>Add 100 &#956;l of cell suspension to microtube containing 10 &#956;g of VAMP2-pHlourin expression plasmid and electroporate with a nucleofector according to manufacturer protocol9.</li>
		<li>After transfections immediately add 500 &#956;l of trypsin quenching media, remove suspension from cuvette and plate cells at a density of 400,000 cells per 35 mm PDL-coated glass bottom dish.</li>
	</ol>
	</li>
	<li>Plate all cortical neurons in 2 ml of serum free media.</li>
</ol>
2. SNARE Complex Formation Assay
Note: SDS-resistant SNARE complexes were processed and analyzed as originally described10 with the modifications detailed below. For validated alternative antibodies to the ones used here, see the materials section.
<ol>
	<li>Stimulate E15.5 cortical neurons 2 days in vitro (DIV) with 250 ng/ml netrin-1 or sham control for 1 hr prior to lysis.</li>
	<li>Prior to processing samples, cool centrifuge to 4 &#176;C, and set water bath temperature to 37 &#176;C, and heat block to 100 &#176;C.</li>
	<li>Remove cell dishes from incubator and place on ice.
	<ol>
		<li>Aspirate media from cells, replace with ~ 2 ml ice cold Phosphate buffered saline (PBS) gently 2 times for 1 - 2 min per wash.</li>
		<li>For this assay, use 2 wells per condition.</li>
		<li>Aspirate PBS from the first well of a condition and replace with 250 &#956;l homogenization buffer. Leave on ice for 5 min, and then using a cell lifter, homogenize cells on ice.</li>
		<li>Aspirate the PBS from the next well of the same condition and add the recently homogenized mixture to the well. This will increase protein concentrations. Repeat for all conditions.</li>
		<li>Upon completion, pipette homogenized solution to a pre-cooled 1.6 ml microtube on ice and add 20% TritonX-100 to reach a final concentration of 1% TritonX-100. Triturate mix 10 times with 1,000 &#956;l pipette while minimizing bubbles.</li>
	</ol>
	</li>
	<li>Incubate tubes on ice for 2 min&#160;to solubilize proteins. Following incubation, centrifuge for 10 min at 6,010 rcf at 4 &#176;C to pellet non-solubilized material. Move lysate supernatant into new chilled 1.6 ml tube on ice.</li>
	<li>Perform protein concentration analysis using Bradford assay11 and dilute samples to a final protein concentration of 3 - 5 mg/ml with remaining homogenization buffer supplemented with 1% TritonX-100 and 5x sample buffer supplemented with B-Mercaptethanol (BME).</li>
	<li>Divide each experimental sample evenly into 2 tubes. Incubate one sample at 37 &#176;C for 30 min&#160;(SNARE complexes), and the other at 100 &#176;C for 30 min (SNARE monomers). Invert tubes intermittently to keep samples in solution.</li>
	<li>Freeze samples at -20 &#176;C until ready to separate via SDS-PAGE. Prior to running samples via electrophoresis using standard techniques, reboil previously boiled samples for 5 min&#160;at 100 &#176;C, but thaw 37 &#176;C samples at RT.</li>
	<li>Run duplicates of samples (30 - 40 &#956;g of protein per sample) on both an 8% and a 15% gel; the 8% provides ideal separation of the complex, whereas the 15% is used to quantify SNARE protein monomers (SNAP-25 ~25kDa, VAMP2 ~18kDa, syntaxin-1 ~35kDa). Maintain power source at 70 V until the dye line is through the stacking gel and increase voltage to 90V. Run gels until the dye runs off.</li>
	<li>To preserve monomers, transfer proteins to 0.2 &#956;m nitrocellulose membrane on ice using cold transfer buffer with 20% Methanol (MeOH) added, at 70 V for 45 min.</li>
	<li>Dry membrane in a covered dish for 2 hr to O/N at RT (O/N provides the best results).</li>
	<li>Block SNARE complex membranes in 10% Bovine Serum Albumin (BSA) for 1 hr at RT.</li>
	<li>Prepare primary antibodies (recognizing specific SNARE complex components) at a dilution of 1:1,000 and probe O/N at 4 &#176;C on a rotary shaker.
	<ol>
		<li>Rinse blots in Tris Buffered Saline plus 0.1% Tween-20 (TBS-T) 3 times for 5 min&#160;each.</li>
	</ol>
	</li>
	<li>Prepare fluorescent secondary antibody solutions at a dilution of 1:20,000 in 1% BSA in TBS-T and probe for 1 hr, covered at RT. Repeat Step 2.12.1 after 1 hr.</li>
	<li>Image blots on a fluorescent scanning machine equipped with software suite and quantify both the SNARE protein complex (immunoreactive bands above 40kDa) and monomer bands (immunoreactive bands at 25 kDa, 18 kDa, 35 kDa for SNAP-25, VAMP2 and syntaxin-1, respectively) using manufacturer&#39;s instructions for drawing rectangular ROIs.</li>
</ol>
3. Imaging Exocytic Events via TIRF Microscopy
Note: This protocol requires specialized microscopy equipment including an environmental chamber to maintain temperature, humidity and CO2, an inverted TIRF microscope equipped with an epifluorescent illumination, a high magnification/ high numerical aperture (NA) TIRF objective, an automated XYZ stage, and a sensitive Charge Coupled Device (CCD) detector. This protocol uses a fully automated inverted microscope equipped with a 100x 1.49NA TIRF objective a solid state 491 nm laser and an Electron Multiplying CCD (EM-CCD). All equipment is controlled by imaging and laser control software. Prior to the beginning the imaging protocol power on the environmental chamber, stage, lamp, computer, and camera.
<ol>
	<li>Select objective within imaging software. Once the objective is in place, fasten the objective heater around the collar.</li>
	<li>Confirm that objective is lowered completely before securing the stage incubator in the stage slot. Pour distilled water into the stage incubator inlets evenly to prevent spill.</li>
	<li>Turn on the incubation system. Open the valve to the CO2 tank and confirm the pressure is appropriate for the system per manufacturer&#39;s instructions. Allow chamber some time to reach 5% CO2 and 37 &#176;C. Prior to adding the imaging dish, place an empty dish in the incubator to avoid water condensation on the objective.</li>
	<li>Power on the laser source.</li>
	<li>Open stage incubator and add immersion oil to the lens. Place sample in incubator and stabilize with stability arms or a dish weight. Raise the objective until the oil makes contact with the bottom of the sample. Switch to transmitted light illumination and find neuronal focal plane through the oculars.</li>
	<li>Start laser software and connect to the laser control software. Set illumination to widefield and select the objective being used (100X 1.49 NA TIRF is recommended). Set refractive index of the sample (cells ~1.38). Adjust laser intensity by unchecking &#34;TTL&#34; for the 491 nm laser. Adjust the slider to 100 then bring it back down to a value between 20 - 40%. Recheck &#34;TTL&#34;.</li>
	<li>Focus on sample again in transmitted light illumination. Go to imaging software and select 491 nm laser illumination and open shutter. Fine adjust the focal point of the laser on the ceiling and center the point to the center of the closed field diaphragm with the condenser removed. Place condenser upside down on the optical bench, so as not to scratch lens.</li>
	<li>Replace condenser and go to TIRF software and set penetration depth (PD) to 110 nm. Switch from widefield illumination to TIRF illumination mode for imaging.</li>
	<li>Find VAMP2-phluorin expressing cells through the oculars using widefield epifluorescence with epifluorescent light source.
	<ol>
		<li>Adjust imaging parameters (exposure time, gain and laser power) to maximize signal to noise ratio and dynamic range using the minimal exposure time and laser intensity to reduce photobleaching and phototoxicity (for example: exposure between 50 - 100 msec, with a 15 - 30 gain at 30% maximum laser power).</li>
		<li>Set continuous autofocus per cell. Acquire a timelapse image set with acquisition occurring every 0.5 sec for 5 min. For the netrin-1 stimulated condition, add 500 ng/ml netrin-1 to dish of cells in a laminar flow hood and return the dish to the incubator for 1 hr prior to imaging.</li>
	</ol>
	</li>
	<li>To quantify the frequency of exocytic events normalized per cell area and time, open image stacks in ImageJ by dragging the file into the window or going to File&#62; Open&#62; Filename (Figure&#160;2A Inset 1).
	<ol>
		<li>To remove stable fluorescent signals that do not represent vesicle fusion events, create an average z projection of the entire stack using Image&#62;Stack&#62;Z-Project&#62;Projection type: Average Intensity. Subtract this mean image from each image in the timelapse using Process&#62; Image Calculator&#62; Image1: your stack Operation: Subtract Image2 newly created average z projection. This emphasizes exocytic events (Figure&#160;2A Inset 2 - 3).</li>
		<li>Count exocytic fusion events by eye. Exocytic events are defined as the appearance of a diffraction-limited fluorescence signal, which rapidly diffuses as VAMP2-phluorin diffuses within the plasma membrane (Figure&#160;2A Inset 6).</li>
		<li>Use the Threshold command to highlight the first image using Image&#62;Adjust&#62;Threshold&#62; adjust slider until the whole cell is threshold highlighted (Figure&#160;2A Inset 7 - 8).</li>
		<li>Under Analyze, choose SetMeasurements and check area and display label. Select the thresholded cellular area using the wand tracing tool and press &#39;m&#39; to measure (Figure&#160;2A Inset 7 - 8).</li>
		<li>Transfer both the exocytic fusion events count, the cell area measurements, and the elapsed imaging time to a spreadsheet to calculate number of exocytic events/mm2/time (Figure&#160;2A Inset 9).</li>
	</ol>
	</li>
</ol>
4. Differential Interference Contrast (DIC) Timelapse Microscopy of Axon Branching
Note: A complete protocol and demonstration for a general approach to DIC imaging is available12. While this protocol utilizes DIC, other transmitted light microscopy methods may be used for the same purposes (for example: phase contrast).
<ol>
	<li>At 2 DIV, place glass bottom imaging dish containing untransfected neurons in pre-warmed environmental chamber to maintain a humid environment with 5% CO2 and 37 &#176;C. Utilize a microscope equipped with a 60X&#160;Plan-Apochromat, 1.4 NA DIC objective lens and high NA condenser for best image quality and resolution.</li>
	<li>Adjust the focal plane to find neurons through the oculars using transmitted light illumination.</li>
	<li>Using the multi area acquisition function on imaging software, find and save the XYZ locations of at least 6 cells. Position the stage so that neurons of interest fit within the field of view for best results.
	<ol>
		<li>Stimulate with 250 ng/ml netrin-1 and press &#39;start multi area acquisition&#39;. Sequentially acquire images at each position every 20 sec for 24 hr, pausing acquisition and refocusing as necessary.</li>
	</ol>
	</li>
	<li>Review images in imaging software using Apps&#62;Review Multi Dimensional Data&#62;open file name. Identify stable axon branches (20 &#181;m long) that form during the imaging session. Use the line draw tool to measure a stableaxon branch from the base at the axon to the tip to ensure the 20 &#181;m length qualification is met.
	<ol>
		<li>In a spreadsheet, record the frame number after netrin stimulation when membrane protrusions initiate in areas where branches later form as well as the frame number after netrin stimulation when nascent branches reach 20 &#181;m in length.</li>
		<li>Multiply the number of frames between protrusion and branch formation by 20 sec to calculate the formation time per branch.</li>
	</ol>
	</li>
</ol>
5. Toxin Manipulations and Fixed Cell Immunofluoresence
<ol>
	<li>At 2 DIV, treat neurons in experimental conditions with 250 ng/ml&#160;netrin-1 and/or 10 nM Botulinum A toxin (BoNTA), which cleaves the SNARE protein SNAP25 and effectively inhibits SNARE mediated exocytosis13, plus 250 ng/ml netrin-1. Leave one set of untreated neurons as control condition. 
	CAUTION: BoNTA requires the use of eye and respiratory protection when preparing and using solutions. Maintain all contaminated materials as biohazardous material and autoclave as soon as possible.</li>
	<li>At 3 DIV (24 hr post treatment) aspirate media, and immediately replace with at least 1 ml of PHEM fix (see Table&#160;1 for details) warmed to 37 &#176;C. Incubate for 20 min&#160;in the dark at RT.</li>
	<li>Rinse 3x with PBS (for future use seal with parafilm and store at 4 &#176;C).</li>
	<li>Place coverslips in dark, humidified staining chamber and permeabilize the cell membranes for 10 min&#160;in freshly diluted 0.2% TritonX-100 in PBS (made from 10% TritonX-100 stock).</li>
	<li>Remove permeabilization solution and rinse with 50 &#181;l of 1x with PBS. Block for 30 min&#160;in ~50 &#181;l of 10% Bovine Serum Albumin in PBS (BSA-PBS) or 10% Boiled Donkey Serum in PBS at RT.</li>
	<li>Rinse with 1x PBS again. Incubate coverslips in 50 &#181;l of primary antibody (1:1,000 &#946;III tubulin, to confirm neuronal identity) in 1% BSA-PBS for 1 hr at RT, in the dark.</li>
	<li>Perform 3x 5 min&#160;washes in PBS. Incubate coverslips in 50 &#181;l of spectrally-distinct secondary antibody for tubulin (1:400), and fluorescent phalloidin (varies by excitation: 488 phalloidin at 1:400, 647 phalloidin at 1:100) in 1% BSA-PBS for 1 hr.</li>
	<li>Wash 3x 5 min&#160;in 1x&#160;PBS and mount coverslips onto slides in mounting media.</li>
	<li>Vacuum excess mounting media from the edges of the coverslip and seal with clear nail polish.</li>
	<li>Collect widefield epifluorescence images on an inverted microscope with a 40X 1.4NA objective, epifluorescent capabilities and EM-CCD.
	<ol>
		<li>Manually analyze branching using ImageJ. Open image stacks in ImageJ by dragging the file into the window or going to File&#62; Open&#62; Filename (Figure&#160;4A inset 1).</li>
		<li>Using line&#62;segmented line, trace the axon, defined as the longest neurite extending from the soma(Figure&#160;4A inset 2 - 3).</li>
		<li>Using Analyze&#62;Tools&#62;ROI Manager, save the axon tracing as a region of interest. Trace and save each axon branch, defined as a neurite &#8805;20 &#181;m in length. Include only primary branches (outgrowths directly sprouting from the axon) in the analysis (Figure&#160;4A inset 3 - 4).</li>
		<li>Press &#39;m&#39; to measure the saved regions of interest, and transfer lengths in pixels to a spreadsheet to calculate the length in &#181;m.</li>
		<li>Divide the number of axon branches &#8805;20 &#181;m in length, by the length of the axon in &#181;m divided by 100 to get number of axon branches per 100 &#181;m length.</li>
	</ol>
	</li>
</ol>

<ol><li>Drachman, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Do+we+have+brain+to+spare%3F%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">Do we have brain to spare?</a> Neurology. 64 (12), 2004-2005 (2005).</li>
<li>Serafini, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Netrin-1+Is+Required+for+Commissural+Axon+Guidance+in+the+Developing+Vertebrate+Nervous+System%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Netrin-1 Is Required for Commissural Axon Guidance in the Developing Vertebrate Nervous System.</a> Cell. 87 (6), 1001-1014 (1996).</li>
<li>Métin, C., Deléglise, D., Serafini, T., Kennedy, T. E., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+netrin-1+in+the+guidance+of+cortical+efferents%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A role for netrin-1 in the guidance of cortical efferents.</a> Development. 124 (24), 5063-5074 (1997).</li>
<li>Kennedy, T. E., Serafini, T., de la Torre, J. R., Tessier-Lavigne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Netrins+are+diffusible+chemotropic+factors+for+commissural+axons+in+the+embryonic+spinal+cord%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.</a> Cell. 78 (3), 425-435 (1994).</li>
<li>Sun, K. L. W., Correia, J. P., Kennedy, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Netrins%3A+versatile+extracellular+cues+with+diverse+functions%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Netrins: versatile extracellular cues with diverse functions.</a> Development. 138 (11), 2153-2169 (2011).</li>
<li>Pfenninger, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plasma+membrane+expansion%3A+a+neuron%27s+Herculean+task%5BTitle%5D)+AND+%22Nature+Reviews+Neuroscience%22%5BJournal%5D)">Plasma membrane expansion: a neuron's Herculean task.</a> Nature Reviews Neuroscience. 10 (4), 251-261 (2009).</li>
<li>Winkle, C. C., McClain, L. M., Valtschanoff, J. G., Park, C. S., Maglione, C., Gupton, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+Netrin-1-sensitive+mechanism+promotes+local+SNARE-mediated+exocytosis+during+axon+branching%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">A novel Netrin-1-sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching.</a> The Journal of Cell Biology. 205 (2), 217-232 (2014).</li>
<li>Viesselmann, C., Ballweg, J., Lumbard, D., Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nucleofection+and+Primary+Culture+of+Embryonic+Mouse+Hippocampal+and+Cortical+Neurons%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons.</a> Journal of Visualized Experiments. (47), e2373(2011).</li>
<li>Miesenböck, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394 (6689), 192-195 (1998).</li>
<li>Hayashi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptic+vesicle+membrane+fusion+complex%3A+action+of+clostridial+neurotoxins+on+assembly%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.</a> The EMBO Journal. 13 (21), 5051-5061 (1994).</li>
<li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72, 248-254 (1976).</li>
<li>Centonze Frohlich, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phase+Contrast+and+Differential+Interference+Contrast+%28DIC%29+Microscopy%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Phase Contrast and Differential Interference Contrast (DIC) Microscopy.</a> Journal of Visualized Experiments. (17), (2008).</li>
<li>Blasi, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Botulinum+neurotoxin+A+selectively+cleaves+the+synaptic+protein+SNAP-25%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25.</a> Nature. 365 (6442), 160-163 (1993).</li>
</ol>

The authors have nothing to disclose.

Axon branching is a fundamental neurodevelopmental process and underpins the vast neuroconnectivity of the mammalian nervous system. Understanding the mechanisms involved in localized plasma membrane expansion is integral to our understanding of both normal and pathological neurodevelopment. The use of a multipronged approach incorporating both population level and single cell level methodologies enhances reproducibility and increases spatial and temporal resolution without compromising population level analysis. At the ...

Recent estimates suggest that the human brain contains 1011 neurons with 1014 synaptic connections1, highlighting the importance of axon branching in vivo. Extracellular axon guidance cues such as netrin-1 guide axons to appropriate synaptic partners and stimulate axonal branching, thereby increasing synaptic capacity2-5. Netrin-1-dependent axonal arborization involves substantial plasma membrane expansion6, which we hypothesized requires delivery of additional membrane components via SNARE complex dependent exocytic vesicle fusion7. 
Investigating the role of SNARE-mediated exocytosis in netrin-1 dependent axon branching is complicated by several factors. First, the heterogeneity of cortical neurons increases the sample size required to identify significant effects, complicating single cell techniques like imaging. Second, although biochemical techniques permit observation of changes that occur at the population level, they lack the temporal and spatial resolution necessary to localize plasma membrane expansion to the axon in the time frame of axon branching. Lastly, although axon branches form over hours, the cellular changes that contribute to axonal extension may begin within minutes and occur on the order of seconds, thus extending the temporal scope for experimental consideration. 
We outline a multi-technique approach that addresses these diverse temporal and spatial scales of exocytosis and axon branching, and thus enhances our understanding of the fundamental cellular mechanisms. Utilizing these approaches provides evidence that supports a critical role for SNARE-mediated exocytosis in axon branching.

<table><tbody><tr><td>6-well tissue culture treated plates</td><td>Olympus Plastics</td><td>25-105</td><td></td></tr><tr><td>glass coverslips</td><td>Fisher scientific</td><td>12-545-81</td><td>12CIR-1.5; must be nitric acid treated for 24 hours, rinsed in DI water 2x, and dried prior to use. Must be coated with 1 mg/ml Poly-d-lysine and rinsed prior to plating cells.</td></tr><tr><td>Amaxa nucleofection solution</td><td>Lonza</td><td>VPG-1001</td><td>100 ml/transfection</td></tr><tr><td>Amaxa Nucleofector/electroporator</td><td>Lonza</td><td></td><td>program O-005</td></tr><tr><td>35 mm Glass bottom live cell imaging dishes</td><td>Matek Corporation</td><td>p356-1.5-14-C</td><td>must be coated with 1 mg/ml Poly-d-lysine and rinsed prior to plating cells</td></tr><tr><td>Olympus IX81-ZDC2 inverted microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Lambda LS xenon lamp</td><td>Sutter Instruments Company</td><td></td><td></td></tr><tr><td>Environmental Stage top incubator</td><td>Tokai Hit</td><td></td><td></td></tr><tr><td>100x 1.49 NA TIRF objective</td><td>Olympus</td><td></td><td></td></tr><tr><td>Andor iXon EM-CCD</td><td>Andor</td><td></td><td></td></tr><tr><td>Odyssey Licor Infrared Imaging System</td><td>LI-COR</td><td>Odyssey CL-X</td><td>Used for scanning blots</td></tr><tr><td>Image studio software suite</td><td>LI-COR</td><td></td><td>Used for scanning on the Odyssey Infrared system; Image studio lite used for offline analysis of blots</td></tr><tr><td>Metamorph for Olympus</td><td>Molecular devices, LLC</td><td>version 7.7.6.0</td><td>Software used for all imaging and the analysis of DIC timelapse</td></tr><tr><td>CELL TIRF control software</td><td>Olympus</td><td></td><td>Software used to control lasers for TIRF imaging</td></tr><tr><td>Fiji (Image J)</td><td>NIH</td><td>ImageJ Version 1.49t</td><td></td></tr><tr><td>60x Plan Apochromat 1.4 NA objective</td><td>Olympus</td><td></td><td></td></tr><tr><td>40x 1.4 NA Plan Apochromat objective</td><td>Olympus</td><td></td><td></td></tr><tr><td>Neurobasal media</td><td>GIBCO</td><td>21103-049</td><td>Base solution for both serum free and trypsin quenching media</td></tr><tr><td>Supplement B27</td><td>GIBCO</td><td>17504-044</td><td>500 ml/50 ml Serum free media and Trypsin Quenching media</td></tr><tr><td>L-Glutamine</td><td></td><td>35050-061</td><td>1 ml/50 ml Serum free media</td></tr><tr><td>Bovine serum albumin</td><td>Bio Basic Incorporated</td><td>9048-46-8</td><td>10% solution in 1x PBS for blocking coverslips; 5% solution in TBS-T for blocking nitrocellulose membranes.</td></tr><tr><td>10x&amp;nbsp; trypsin</td><td>Sigma</td><td>59427C</td><td></td></tr><tr><td>HEPES</td><td>CELLGRO</td><td>25-060-Cl</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (DPBS)+ Ca + Mg</td><td>Corning</td><td>21-030-cm</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Corning/CELLGRO</td><td>35-010-CV</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Corning/CELLGRO</td><td>20-021-CV</td><td></td></tr><tr><td>NaCl</td><td>Fisher scientific</td><td>BP358-10</td><td></td></tr><tr><td>EGTA</td><td>Fisher scientific</td><td>CAS67-42-5</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher scientific</td><td>BP214-500</td><td></td></tr><tr><td>TRIS HCl</td><td>Sigma</td><td>T5941-500</td><td></td></tr><tr><td>TRIS base</td><td>Fisher scientific</td><td>BP152-5</td><td></td></tr><tr><td>N-Propyl Gallate</td><td>MP Biomedicals</td><td>102747</td><td></td></tr><tr><td>Glycerol Photometric grade</td><td>Acros Organics</td><td>18469-5000</td><td></td></tr><tr><td>Glycerol (non optics grade)</td><td>Fisher scientific</td><td>CAS56-81-5</td><td></td></tr><tr><td>B-mercaptoethonal</td><td>Fisher scientific</td><td>BP176-100</td><td></td></tr><tr><td>SDS</td><td>Fisher scientific</td><td>BP166-500</td><td></td></tr><tr><td>Distilled Water&amp;nbsp;</td><td>GIBCO</td><td>152340-147</td><td></td></tr><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>p-7886</td><td>Dissolved in sterile water at 1 mg/ml</td></tr><tr><td>Botulinum A toxin BoNTA</td><td>List Biological Laboratories</td><td>128-A</td><td></td></tr><tr><td>Rabbit polyclonal anti human VAMP2</td><td>Cell signaling</td><td>11829</td><td></td></tr><tr><td>Mouse monoclonal anti rat Syntaxin1A</td><td>Santa Cruz Biotechnology</td><td>sc-12736</td><td></td></tr><tr><td>Goat polyclonal anti human SNAP-25</td><td>Santa Cruz Biotechnology</td><td>sc-7538</td><td></td></tr><tr><td>Mouse monoclonal anti human &amp;beta;III-tubulin&amp;nbsp;</td><td>Covance</td><td>MMS-435P</td><td></td></tr><tr><td>Alexa Fluor 568 and Alexa Fluor 488 phalloidin, or Alexa Fluor 647</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>LI-COR IR-dye secondary antibodies</td><td>LI-COR</td><td>P/N 925-32212,P/N 925-68023, P/N 926-68022</td><td>800 donkey anti-mouse, 680 donkey anti rabbit, 680 donkey anti goat</td></tr><tr><td>0.2 &amp;#956;m pore size nitrocellulose membrane</td><td>Biorad</td><td>9004-70-0</td><td></td></tr><tr><td>Tween-20</td><td>Fisher scientific</td><td>BP337-500</td><td></td></tr><tr><td>Methanol</td><td>Fisher scientific</td><td>S25426A</td><td></td></tr><tr><td>Bromphenol Blue</td><td>Sigma</td><td>B5525-5G</td><td></td></tr><tr><td>Sucrose</td><td>Fisher scientific</td><td>S6-212</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher scientific</td><td>O-4042-500</td><td></td></tr><tr><td>Triton-X100</td><td>Fisher scientific</td><td>BP151-500</td><td></td></tr><tr><td>TEMED</td><td>Fisher scientific</td><td>BP150-20</td><td></td></tr><tr><td>40% Bis-Acrylimide</td><td>Fisher scientific</td><td>BP1408-1</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Alternative Validated Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mouse Monoclonal Anti-Syntaxin HPC-1 clone</td><td>Sigma Aldrich</td><td>S0664</td><td></td></tr><tr><td>&amp;nbsp;Mouse Monoclonal Synaptobrevin 2 (VAMP2)</td><td>Synaptic Systems</td><td>104-211</td><td></td></tr><tr><td>Mouse Monoclonal SNAP25</td><td>Synaptic Systems</td><td>111-011</td><td></td></tr></tbody></table>

utilizing combined methodologies to define the role of plasma membrane delivery during axon branching and neuronal morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Utilizing in vitro biochemical techniques to assay the amount of SDS-resistant SNARE complexes in a population of neurons. Figure 1 shows the resulting western blot following completion of the SDS-resistant SNARE complex assay probed for SNAP-25, syntaxin1A and VAMP2.
TIRF microscopy at the basal cell membrane provides high resolution images of individual exocytic fusion events in single cells. ...

Watch this Scientific Journal Video about Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis at JoVE.com

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

utilizing-combined-methodologies-to-define-role-plasma-membrane

Research

JoVE Journal

Neuroscience

6.1K Views.  The University of North Carolina at Chapel Hill. Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change. 

Video: Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Biology

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to bridge the lesion site, properly organized axonal alignment is required. Since demyelination after nerve injury strongly impairs the conductive capacity of surviving axons, remyelination is critical for successful functioning of regenerated nerves. Previously, we demonstrated that mesenchymal stem cells (MSCs) aligned on a pre-stretch induced anisotropic surface because the cells can sense a larger effective stiffness in the stretched direction than in the perpendicular direction. We also showed that an anisotropic surface arising from a mechanical pre-stretched surface similarly affects alignment, as well as growth and myelination of axons. Here, we provide a detailed protocol for preparing a pre-stretched anisotropic surface, the isolation and culture of dorsal root ganglion (DRG) neurons on a pre-stretched surface, and show the myelination behavior of a co-culture of DRG neurons with Schwann cells (SCs) on a pre-stretched surface.

This protocol describes the isolation of dorsal root ganglion (DRG) neurons isolated from rats and the culture of DRG neurons on a static pre-stretched cell culture system to enhance axon alignment, with subsequent co-culture of Schwann Cells (SCs) to promote myelination.

Axon regeneration is a chaotic process due largely to unorganized axon alignment. Therefore, in order for a sufficient number of regenerated axons to ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Visualization of Thalamocortical Axon Branching and Synapse Formation in Organotypic Cocultures

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) axons form branches and synapses in specific layers of the cerebral cortex. Despite the obvious spatial correlation between axon branching and synapse formation, the causal relationship between them is poorly understood. To address this issue, we recently developed a method for simultaneous imaging of branching and synapse formation of individual TC axons in organotypic cocultures.
This protocol describes a method which consists of a combination of an organotypic coculture and electroporation. Organotypic cocultures of the thalamus and cerebral cortex facilitate gene manipulation and observation of axonal processes, preserving characteristic structures such as laminar configuration. Two distinct plasmids encoding DsRed and EGFP-tagged synaptophysin (SYP-EGFP) were co-transfected into a small number of thalamic neurons by an electroporation technique. This method allowed us to visualize individual axonal morphologies of TC neurons and their presynaptic sites simultaneously. The method also enabled long-term observation which revealed the causal relationship between axon branching and synapse formation.

This protocol describes a method for simultaneous imaging of thalamocortical axon branching and synapse formation in organotypic cocultures of the thalamus and cerebral cortex. Individual thalamocortical axons and their presynaptic terminals are visualized by a single cell electroporation technique with DsRed and GFP-tagged synaptophysin.

Axon branching and synapse formation are crucial processes for establishing precise neuronal circuits. During development, sensory thalamocortical (TC) ...

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. ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

Samenvatting

Meer video's verkennen