The goal of this experiment is to extend an axon while accurately measuring forces. This is achieved by making a directly calibrated wire reference needle following, wire reference needle calibration, calibrated, intermediate, and working needles are prepared in the desired force range. Next growth cones are towed with a working needle in order to elongate an axon.
The resulting axonal elongation is recorded and forces are measured based on the flexing of the calibrated needle. This method can answer key questions about the biophysics of axon elongation. Begin this procedure by using an adjustable microneedle puller and glass capillary tubes to fabricate needles with tapered tips about four millimeters in length.
Store the needles in a covered 160 millimeter Petri dish. Next, make a wire calibration needle by inserting a 0.001 inch chromal wire through the inside of a glass needle with its tip broken off. Pull the wire out of the tip to a length of 26 millimeters and glue it in place with super glue.
Bend the distal one millimeter of the wire into a hook. Then micro weights are made from one meter of 0.003 inches constant in wire. To measure the wire, tape it to a meter stick.
To assist in getting it as straight as possible. Weigh the one meter wire and accurately cut it into one centimeter pieces. Bend several pieces of the wire into vase.
Calibrating the wire needles requires a dissecting microscope with a measuring vertical and a micro manipulator. The microscope is tilted 90 degrees on its side. Using a universal boom stand, so the downward deflection of the wire needle can be observed.
Mount the wire needle in a tool holder on a micro manipulator so it is perpendicular to the optical axis of the microscope. Line up the hook of the wire needle with a radical mark. Hang the one centimeter micro weight on the hook and record the deflection.
The bending constant of the reference wire is equal to the mass of the micro weight divided by the observed deflection. After calibrating, prepare the needle for the next step by cutting off the hook at the way waypoint with the razor blade to calibrate the intermediate glass needles. An inverted microscope with an ocular redle is required.
On one side of the microscope stage is a mechanical microm manipulator used to hold the wire needle. On the other side is a hydraulic microm manipulator used to move the intermediate glass needle be in calibrated using the microm manipulators bring both needles together in the center of the optical field at a 45 degree angle in a 160 millimeter by 30 millimeter Petri dish of water in a 10 x field. Next, perform a series of deflections for each of these deflections.
Note the initial amount deflected and the final zero load position of the glass needle after release. This creates 10 pairs of data points. To prepare the 10 data pairs for a linear regression, the initial deflection is subtracted from the second number of the recorded pairs.
Add five control pairs of zero zero to the 10 experimental data pairs to anchor the linear regression at zero, which is zero deflection. At zero force. Calculate the linear regression with these 15 data pairs.
The spring constant of the intermediate glass needle is equal to the wire needles known bending constant divided by the slope of the regression line. Then calibrate the working glass needles against the intermediate glass needle as just described. Glass needles of less than two microns per micrometer may be slightly shortened to bring them into an appropriate stiffness range.
Press the needles to be coated into the side of a lump of clay held above a 10 milliliter beaker of the attachment factor solution such that only the needle tip is immersed in the solution sequentially. Dip the needles in 0.1%polylysine solution for 30 minutes. Then dip the needles in conval and a for 30 minutes while treating the needle with attachment factors.
Turn on the incubator or microscope stage heating system. So the stage will be at thermal equilibrium. The towing workstation on a vibration isolation table consists of an inverted microscope fitted with a hydraulic manipulator holding a counterbalanced custom extension into which the double tool holder is inserted.
Insert an uncalibrated reference needle into a needle holder so that it extends as long as possible. Place this assembly in the right side of the double tool holder. Then insert the treated working needle into a needle holder so it extends half as far as the reference needle and place it in the left side of the double tool holder.
Preset the hydraulic micro manipulator so that the vertical control is all the way up. Then bring the two needle tips together by using the side to side adjusting screw. Position the needle.
So the tips are even examine the pair from the side by eye and use the right side up down screw to bring the tips into the same plane. Swing the tool holder and place the needle tips into the light path above the surface of the media. Ling them down steeply.
Tighten the ball joint screw at the base. Focus upward above the cells on the bottom of the dish viewing with the 10 x objective. Lower the needles into the media to find the needle tips in the microscope's visual field.
Before bringing them too far down into the culture dish. Move the needle side to side backward or forward and look for their shadow. Once a needle is in view, turn the side to side screw to see which needle is found.
If it moves with the screw, it is the reference needle. If it doesn't, it's the calibrated needle. When both needles are located, use the fine controls of the double tool holder to line them up.
The objective is to have the needle tips about 50 micrometers apart or about an inch on the monitor. The reference should be farther above the culture dish and therefore somewhat out of focus. When the towing needle is in focus on the dish record the image of this final adjusted position.
The separation of the needles is the zero reference distance. Raise the needles above the specimen plane and park them at the edge of the visual field. Choose an experimental axon using the guidelines in the written protocol.
Move the needles close to the growth cone of the axon and record the zero distance again. Next, air up the vibration isolation table to manipulate a growth cone onto the needle. Place the towing needle beside the growth cone below it, and then lower the needle against the dish bottom.
This will cause the needle to deflect up, bending and sliding along the dish into the growth cone, and then dislodging the growth cone and moving it upward. Press the needle against the dish bottom just enough so that the growth cone hooks around the needle tip, but not so far that it slips over the needle and slides backward. Wait 20 minutes to let the grip become established.
When the growth cone is on the needle, put a slight tension on it By moving the needle to the right and slightly raising the needle, compensate the downward movement of the needle by moving it a bit forward as it is raised. Raise the needle in stages until the attached process is extending perpendicular from the needle and above the surface of the dish. Towing is accomplished by moving the needles away from the cell body.
In small increments, forces are observed as an increasing gap between the needles while gradually increasing the tension. Carefully observe for any signs of detachment of the growth cone from the needle. Maintain the media level in the dish by adding water hourly to counterbalance evaporation.
Using a mark on the side of the dish to indicate the initial depth with a long past your pipette slowly add water at the side of the dish away from the needles at the end of an experiment. Before lifting the needles out of the dish, recheck the zero distance. Read the positions of the needles along the horizontal axis on the screen.
Also record the position of the cell body and the times the separation distance of the needles minus the unloaded zero distance multiplied by the bending constant for the needle equals the microns of force being applied.Shown. Here is an example field of cells with several possible choices for controlling axonal development. The horizontal exon with a large growth cone is a good selection.
The cell body is positioned near the edge of the screen and the needles are brought in close to the growth cone. An image is taken of the unloaded needle separation, which is the zero distance. Before the experiment begins, the towing needle is set against the growth cone to see if a spontaneous attachment can be achieved.
Then the needle is pushed into the growth cone a little more. The push up maneuver is performed by lowering the needle against the dish bottom, causing the needle to bend and slide into the growth cone dislodging it. A small increment of forward tension has also been added.
After 15 minutes, the axon looks like it has extended with the forward motion of the needle. Next, more tension is added to the towing needle and the raising of the needle begins. Additional tension is added shown by the increased separation of the needles.
At this point, the axon is clearly extending. The reference needle has not moved forward, but the axon has grown toward it, lessening the tension. Also, the needles have been raised slightly.
Here still, more tension is added to the towing needle. Significant growth has occurred in response to the tension loaded in the flexed towing needle. At the end of the experiment, a final zero check is performed.
The entire process is demonstrated in this time-lapse sequence. After watching this video, you should have a good understanding of how to calibrate needles, manipulated growth cone onto a needle, and measure the forces while extending an axon.
