The overall goal of this procedure is to make quantifiable observations of the temporal and spatial occurrence of neuronal exocytic vesicle fushion and axon branching. This method can help answer key questions in the neuroscience field, such as when and where vesicle fusion inserts membrane material into the expanding plasma membrane during neuronal development. The main advantage of this technique is that it combines live cell imaging to acquire both high spatial and temporal resolution in single cells and biochemical approaches to acquire population based information.

After culturing and simulating cortical neurons with Netrin-1 or a control according to the protocol text. Aspirate PBS from the first well of a condition and replace with 250 microliters of homogenization buffer. Incubate on ice for five minutes, and then using a cell lifter, homogenize the cells on ice.

Aspirate the PBS from the next well of the same condition and add the recently homogenized mixture to the well which will increase protein concentrations. Next, transfer the homogenized solution to a pre-cooled 1.6 milliliter microfuge tube on ice, and add 20%Triton X-100 to reach a final concentration of 1%Then with a 1, 000 microliter pipette, triturate the mixture ten times while minimizing bubbles. Incubate the tubes on ice for two minutes to solubilize the proteins.

Then centrifuge at 6, 010 RCF at 4 degrees Celsius for 10 minutes to pellet the non-solubilized material. Transfer the lie state supernatant into a new chilled 1.6 milliliter tube on ice. Next, use the Bradford assay to determine protein concentration.

Then, use homogenization buffer with 1%Triton X-100 and 5X sample buffer supplemented with BME to dilute the samples to a final concentration of 3-5 milligrams per milliliter. Divide each experimental sample evenly into two tubes. Then incubate one sample at 37 degrees Celsius for 30 minutes, and the other at 100 degrees Celsius for 30 minutes.

Invert the tubes intermittently to keep samples in solution. Then carry out SDS page and Western blotting according to the text protocol. After setting up the TIRF microscope and the sample and finding a neuronal focal plane according to the text protocol.

Start the laser software and connect to the laser control software. Set the illumination to wide field and select the objective. Then set the refractive index of the sample and adjust the laser intensity by unchecking TTL for the 491 laser.

Imaging parameter optimization is important during imaging of floor intact exocytic vesicles to avoid focus drift and phototoxicity while producing images of high signal to noise ratio sufficient for analysis. Adjust the slider to 100 and then bring it back down to a value between 20 and 40. Then recheck TTL.

Next, focus on the sample again in transmitted light illumination. Then in the imaging software, select the 491 laser illumination and open the shutter. Place the condenser upside down on the optical bench so as not to scratch the lens.

Fine adjust the focal point of the laser on the ceiling and with the condenser removed, center the point to the center of the closed field diaphragm. Then replace the condenser and in the TIRF software sent the penetration depth to 110 nanometers. Then switch from wide field illumination to TIRF illumination mode for imaging.

Now, using wide field epifluorescence find VAMP2 fluorent expressing cells through the oculars. Then to reduce photobleaching and phototoxicity, adjust the imaging parameters to maximize the signal to noise ratio and dynamic range using minimal exposure time and laser intensity. Set continuous auto focus per cell.

Then acquire a time lapse image set with acquisition occurring every 0.5 seconds for five minutes. For the Netrin-1 stimulated condition in a laminar flow hood, add to a final concentration of 500 nanograms per milliliter of Netrin-1 to a dish of cells and return the dish to the incubator for one hour prior to imaging. To quantify the frequency of exocytic events normalized per cell area and time.

In ImageJ, open image stacks by dragging the file into the window or by choosing File, Open, file name. To remove stable fluorescent signals that do not represent vesicle fusion events, use Image, Stack, Z Project, Projection type Average Intensity. To create an average Z Projection of the entire stack.

Subtract the resulting mean image from each image in the timelapse using Process, Image Calculator, Image 1 your stack, Operation Subtract image two. Resulting in a newly created Z Projection. This will emphasize exocytic events.

Now by eye, count exocytic fushion events, which can be identified as spots of diffraction limited fluorescent signals that rapidly diffuse as VAMP2 fluoren diffuses within the plasma membrane. Highlight the first image with Image, Adjust, Threshold, and adjust the slider until the entire cell is threshold highlighted. Then under Analyze, choose Set Measurements and check the area and display label.

With the wand tracing tool, set the thresholded cellular area and press M to measure. Transfer the collected data to a spreadsheet according to the text protocol. At two days in-vitro, place the glass bottom imaging dish containing untransfected neurons in a pre-warmed humid environmental chamber.

Utilize a microscope equipped with a six DX plan apochromat 1.4 NA DIC objective lens and a high numerical aperture condenser for the best image quality and resolution. Next, with transmitted light illumination adjust the focal plane to find neurons through oculars. For DIC imaging of axon branching, proper setup of cooler illumination will be integral for image optimization and is critical to producing analyzable results.

Then, with the neurons of interest within the field of view, use the multi-area acquisition function in the imaging software to find and save the XYZ locations of at least six cells. Now, add to a final concentration of 250 nanograms per milliliter of Netrin-1 or other factor of interest to simulate the cells and press start multi-area acquisition. Sequentially acquire images at each position every 20 seconds for 24 hours.

Pausing acquisition and refocusing as necessary. Within the imaging software, review images by choosing Apps, Review Multi Dimensional Data and open the desired file. Identify stable axon branches that formed during the imaging session.

Use the trace region tool to measure a stable axon branch from the base of the axon to the tip. After treating neurons with Netrin and botulinum toxin and fixing and immunostaining, and immunostaining for beta-3 tubolin and actin according to the text protocol. Use an inverted microscope to collect wide field epifluorescence images.

Open an image stack in ImageJ and to manually analyze the branching use line, segmented line and trace the axon which is defined as the longest neurite extending from the soma. With Analyze, Tools, ROI Manager, save the axon tracing as a region of interest. Then trace and save each axon branch, which is defined as a neurite greater than or equal to 20 micrometers in length.

Include only primary branches in the analysis. Shown here is a Western blot following the completion of the SDS resistant SNARE complex assay. Probed for SNAP25, Syntaxin 1A, and VAMP2.

SNARE proteins in complex, an identifiable monomers are shown here. An example of a VAMP2 fluroen mediated exocytic event occurring over time in a cortical neuron is shown here. The zoomed insets denote the soma, an axon branch, and an axonal growth cone showing the spatial utility of this assay.

Circles detonate single exocytic fusion events, which can be seen via TIRF microscopy. This time lapse DIC imaging shows the Netrin stimulated formation of an axon branch in real time. These locations denote the initial protrusion from a branch site.

These represent fully formed stable branches of at least 20 micrometers measured from the main axon to the branch tip. In this experiment, cortical neurons at 3 DIV were either treated with Netrin-1, which stimulates branching or botulinum A toxin, which cleaves SNAP25, a component of the SNARE complex and blocks exocytosis. The results demonstrate that SNARE mediated exocytosis is requisite for Netrin dependent branching.

Once mastered, the DIC imaging can be completed in a single overnight imaging session. TIRF imaging of individual exocytic events can be managed in 4-5 hours. The SNARE complex formation biochemistry protocol can be performed in two hours.

While attempting these procedures, it's important to remember to pace yourself and not rush any single step. Each procedure requires attention and patience for the best results. Following this procedure, other methods like cotransfection of tagged vesicle components with tagged candidate cargos can be performed to answer additional questions.

Like what is the cargo of the exocytic vesicles? After it's development, this technique paved the way for researchers in the field of neuroscience to explore neuritogenesis, axon branching, and other stages in morphogensis in any dissociated neuronal culture. After watching this video, you should have a good understanding of how to observe and quantify the spatial and temporal nature of neuronal exocytic vesicle fusion events corresponding to changes in neuronal morphogensis.

Don't forget that working with botulinum toxin or high powered lasers can be extremely hazardous and precautions such as wearing proper safety garments and using the microscope safety lock mechanism should always be taken while performing this procedure.