This article describes a method for inducing axonal varicosities in neurons using a micropipette and fluid pressure. The procedure involves precise positioning of the micropipette and application of a salt buffer to stimulate the neurons.
Begin by securing a micropipette with attached tubing to a micromanipulator.
Attach the other end of the tubing to a syringe filled with a salt buffer via a connector with a turnable valve.
Set the syringe at a height that provides sufficient pressure to induce varicosities in axons.
Axonal varicosities are bead-like swellings that occur along the length of an axon.
Position the micropipette at the desired angle relative to the culture dish.
Use fluorescent light from the microscope to align the micropipette tip and lower it near the surface of the cell culture dish containing salt buffer.
Now, turn on the transmitted light and turn off the fluorescent filter.
Place a coverslip with neurons onto the dish.
First, focus on the micropipette and adjust its position.
Then, focus on the region containing the target neurons.
Open the valve to apply fluid pressure. This micromechanical stimulus induces varicosities in these neurons.
In this procedure, insert the pipette into the screw top of the micromanipulator to hold it into place. Angle the pipette at a 45-degree angle with the surface to the cell culture dish. Turn on the fluorescence filter. Open the aperture, and ensure a fluorescent spot can be seen coming through the objective.
Using the micromanipulator, move the pipette into a position that lines up the tip with the fluorescent spot, and lower the pipette into a position just above the height of the cell culture dish. Once it is in position, turn on the transmitted light and turn off the fluorescence. Using tweezers, transfer one coverslip with the cells facing toward the pipette from the cell culture plate to the cell culture dish containing 2 milliliters of Hank's buffer at room temperature.
Using the fine focus knob and 20-fold objective, focus on a plane about four full turns above the cells. Subsequently, use the micromanipulator to position the pipette tip so that it is focused and in the center left-hand side of the plane, as seen through the eyepieces. Once the pipette is in position, focus the microscope on a plane that contains the region of interest.
In the imaging of 7DIV, GFP-transfected mouse hippocampal neurons showed an axon pre- and post-puffing.
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