Single Cell Electroporation in Organotypic Brain Slice Cultures: An In Vitro Technique to Deliver Plasmids Inside Targeted Neurons in Brain Hippocampal Slices

Begin electroporation by taking a glass pipette containing the desired plasmid solution. Attach it to a micropipette holder fitted with a silver wire electrode, ensuring that the electrode is inserted into the micropipette. Take an electroporation chamber containing an ultrathin hippocampal section submerged in artificial cerebrospinal fluid.

Dip the ground electrode into the fluid present in the chamber for subsequent electroporation. Lower the pipette tip into the electroporation chamber and record the baseline resistance. Use a microscope to position the pipette tip near the target cell. Once the pipette is in contact with the neuronal membrane, its resistance increases sharply.

Apply positive air pressure to create a small invagination in the neuronal membrane. Rapidly apply mild suction to push the membrane up, creating a loose seal between the pipette tip and the membrane. Repeat this process a few more times to condition the membrane and avoid cell lysis.

After conditioning, apply strong suction to form a tight seal with a membrane called gigaohm seal. Use electric pulses to form transient pores in the sealed area of the membrane. These openings facilitate the entry of plasmids inside the targeted neuron. Post electroporation, the pores reseal, entrapping plasmids inside the neuron.

To prepare slice cultures for electroporation, transfer the culture inserts of interest into individual 3-centimeter Petri dishes loaded with 900 microliters of culture medium and place the cultures in a tabletop carbon dioxide incubator. Next, pre-incubate fresh culture inserts with 1 milliliter of slice culture medium per insert in a 3.5-centimeter Petri dish for at least 30 minutes, and sterilize the lines of the electroporation rig with 10% bleach for 5 minutes.

At the end of the perfusion, rinse the lines with the ionized autoclaved water for at least 30 minutes before perfusing with filter-sterilized aCSF containing 0.001 millimolar tetrodotoxin. Set the electroporator pulse to an amplitude of minus 5 volts, a square pulse, a train of 500 milliseconds, a frequency of 50 Hertz, and a pulse width of 500 microseconds. Fill a glass pipette with 5 microliters of plasmid-containing internal solution and gently flick and tap the tip multiple times to remove any trapped bubbles.

Use a dissection microscope to confirm that the tip is not damaged and securely attach the pipette tip to the electrode. When the tip makes contact with the aCSF, record the pipette resistance readout of the electroporator. To isolate a slice culture, cut the culture insert membrane with a sharp blade and use sharp-angled forceps to carefully transfer the slice culture to the electroporation chamber. Then, fix the culture position with a slice anchor.

For electroporation of the cells of interest, apply positive pressure to the pipette by mouth and use the three-dimensional knob controls to maneuver the pipette tip to near the surface of the slice culture. Viewing the culture with the microscope, approach the target cell with the tip, keeping the positive pressure applied until a dimple forms on the cell surface. Upon visualization of the dimple, quickly apply mild negative pressure by mouth so that a loose seal forms between the pipette tip and the plasma membrane.

The membrane will slightly enter the pipette. An approximately 2.5x increase in the initial resistance of the pipette should be observed, as indicated by an increase in tone from the speakers. Quickly reapply positive pressure so that the dimple reforms. Then, immediately complete at least two more pressure cycles as just demonstrated without pausing. After the last pressure pulse, hold the negative pressure for 1 second. Tone from the speakers reaches a stable apex in pitch. Use the foot pedal to quickly pulse the electroporator.

After the electroporation, gently retract the pipette approximately 100 microns from the cell without applying pressure and reapply positive pressure, verifying that the resistance is similar to the baseline readout before approaching the next cell. When all of the cells of interest have been electroporated, transfer the slice culture into one of the prepared fresh culture inserts and place the insert at 35 degrees Celsius for up to 3 days.