In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

How axons navigate the complex central nervous system matrix is a fundamental question in neurobiology. This protocol enables the study of axon growth and growth cone dynamics in the physiological environment with high resolution imaging. This protocol describes a user friendly end to end pipeline for the delivery of DNA in the mouse embryonic brain, preparations for high quality organotypic slices and a step by step guide for image acquisition and analysis.

Although originally designed to study axon dynamics in the embryonic central nervous system, this protocol could be adjusted to allow for organotypic culture and visualization of axon plasticity after traumatic or pathological conditions. The protocol itself is relatively straightforward. However, achieving its first labeling and preservation of brain structures are critical points.

Therefore, the electroporation, brain dissection, and slicing steps require extra care. Demonstrating the procedure will be helpful, and doctoral student is from Bracket's laboratory. After anesthetizing a pregnant female mouse, make an incision to pull out both uterine horns using a cotton bud soaked in warm saline or forceps, carefully grabbing the spaces between the embryos.

Then place the embryos on wet gauze. After cutting open the uterine sac, remove each embryo. Place the embryos in a 10 centimeter dish containing HBSS supplemented with glucose on ice.

For ex utero electroporation, pick up an embryo and place it in the holder. Then carefully insert the glass capillary containing the DNA fast green mix through the embryo skull into the lateral ventricle, and inject two to three microliters of the DNA plasmid mix into each ventricle. Next, hold the embryo's head between platinum tweezer electrodes at the appropriate angle to target the desired brain area, with the cathode facing the area where the DNA transfer is intended.

For in utero electroporation, after pulling out the uterine horns and placing the embryos on a wet gauze as demonstrated previously, use fingertips to gently rotate the embryo inside the uterus until the lambdoidal and saggital sutures are located. Then carefully insert the glass capillary containing the DNA fast green mix through the uterine wall and embryo skull into the lateral ventricle, and inject two to three microliters of the DNA plasmid mix into either one or both ventricles as desired with a maximum of two microliters per ventricle. After the injection, hold the embryo's head between platinum tweezer electrodes at the appropriate angle to target the desired brain area with the cathode facing the area where the DNA transfer is intended.

Once all the required embryos have been electroporated, use a saline soaked cotton bud to gently place the uterine horns back inside the abdominal cavity. Suture the muscle and skin incisions using 5-0 suture material, then secure the wound using suture clips, and disinfect the wound by spraying it with betadine. Place the mouse back in the recovery cage and maintain warmth using a far infrared warming light for at least 20 minutes postprocedure.

Fix the embryo's head under a dissection microscope. Then remove the skin in skull by cutting along the midline starting from the base of the head toward the nose. Peel the skin in skull laterally making a big enough gap for the brain to be excised.

Next, to remove the brain insert the closed tip of sterile dissection scissors starting under the olfactory bulb and moving toward the brain stem. Then cut off the brain stem and trim many loose pieces of meninges around the brain. Using a perforated spoon, pick up the brain and remove the excess liquid by dabbing the bottom of the spoon against dry tissue paper.

Then place the brain in an agarose dish on ice. Next, using a smaller spoon mix the agarose for 10 seconds for even cooling, then maneuver the brain to the middle of the dish placing it horizontally with the dorsal side up and ensuring that it is completely covered with agarose from all directions. Gently pick up the agarose block from the Vibratome workstation and dry the bottom by dabbing against tissue paper.

Then place the block on the glued area of the specimen holder with the rostral side of the brain facing up, and put the specimen holder on ice. Allow the glue to dry for one minute. Cut the brain in coronal slices at an angle of 15 degrees.

Then using clean spatulas, collect the brain slices and place them on a PTFE membrane immobilized in a 35 millimeter glass bottom dish using Parrafin. Collect up to five brain slices per membrane. Next, using a 200 microliter pipette remove the excess HBSS glucose solution from around the slices on the membrane leaving the slices semi dry, then add 500 microliters of prewarmed slice media directly to the space under the membrane and incubate the slices at 35 degrees Celsius with 5%carbon dioxide.

For imaging axon growth, locate a cortex region with low to medium cell density. While for imaging growth cone dynamics, locate a growth cone in the cortex's immediate zone or subventricular zone. Next define a Z stack size.

For axon growth in a large Z stack, set a step size of two micrometers and for growth cones in a smaller Z stack, set a step size of one micrometer. For data analysis, open the image file in Fiji by clicking file, then open, and selecting the image. Obtain the maximum intensity projection of the time lapse by clicking on image, followed by stacks, Z projection, and maximum intensity projection.

Go through the time lapse and locate a growing axon. Once located, draw a line through the growing axon, starting from the tip of the axon in the first frame and following the axon through the entire time lapse. Next, sing the plugin kymo re-slice wide.

Set the scale of the kymograph by going to image, then properties. After setting the distance in micrometers, and pixel width and the time in seconds or minutes, and pixel height, go to analyze and click measure. To measure the volume of the growth cone, open the image file in the image analysis software by clicking file, open, and selecting the file of interest.

Then, select the add new surfaces wizard in step one, under algorithm settings, select segment only a region of interest, and in step two, crop the frame to fit the entire growth cone in all frames. Next in step three, keep the thresholding to absolute intensity and in step four, ensure the entire growth cone region is thresholded. Then in step five, under filter type select number of voxels LMG to one.

Select the execute button to perform all the creation steps and terminate the add new surfaces wizard. Finally, in the statistics tab at the top of the wizard window, select specific values and volume under the detailed tab. Typically, successfully cultured brain slices derived from either in utero or ex utero electroporation show normal cellular distribution and an organized array of radial glia with apically oriented pilo-contacting processes.

Occasionally an ex utero electroporation marked disturbances in the radial glial scaffolding and cultured brain slices are observed making this control staining recommendable. A representative pyramidal cortical projecting neuron expressing Lyn-mNeonGreen and the dynamic behavior of its growth cone is shown here. Additionally, neurons were labeled using a plasmid expressing actin probe to analyze actin dynamics of axonal growth cones in situ.

In situ experiments were also performed with a dual cre dre fluorophore expressing plasmid design, where tRFP or ZsGreen fluorophores could be specifically and individually activated by either dre or cre recombinases, respectively in neighboring neurons. From kymographs, dynamic growth parameters such as growth speed for several axons and growth cone volume over time are easily obtained. This can be used to evaluate the speed of actin treadmilling and the balance between filopodia and lamellapodia during growth cone exploring activity.

It's important to maintain brain structure and fine tune plasmid concentrations to achieve sparse labeling. This is crucial for accurate visualization of axons and growth cones in the cortex. Depending on the selected plasmas and genetic background, this protocol permits users to modify neurons or the environment in which they grow allowing for a wide range of studies.

By enabling high resolution access to neurons in situ, this technique enables neuroscientists to interrogate the dynamic interaction between growth cones and the central nervous system metrics both at morphological and molecular levels.