Neuroscience
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Single-Cell Electroporation across Different Organotypic Slice Culture of Mouse Hippocampal Excitatory and Class-Specific Inhibitory Neurons
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Summary October 6th, 2020
Presented here is a protocol for single-cell electroporation that can deliver genes in both excitatory and inhibitory neurons across a range of in vitro hippocampal slice culture ages. Our approach provides precise and efficient expression of genes in individual cells, which can be used to examine cell-autonomous and intercellular functions.
Transcript
Gene transfection is a vital approach for neurobiology studies. Our electroporation technique enables us to transfect gene of interest interneurons in organotypic hippocampus slice culture with no detectable side effects. This protocol has a much higher transfection efficiency compared to other single cell protocols, but it is still relatively inexpensive and simple to perform.
This technique will be useful for researchers interested in understanding specific molecular and physiological functions of neurons, including cell autonomous mechanisms and transsynaptic protein interactions. To prepare slice cultures for electroporation, transfer the culture inserts of interest into individual three 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 one 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 five 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 five 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 five 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 one second.
When 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 three days.
In this representative experiment, EGFP was transfected into five to 20 pyramidal neurons in the hippocampal CA1 area across seven, 14, and 21 days in vitro slice culture ages. Parvalbumin and vesicular glutamate type 3 interneurons can also be successfully electroporated within the hippocampal CA1 area. Interestingly, transfection is not significantly affected by the day in vitro slice culture age, and does not differ with the transfection efficiency observed in CA1 pyramidal neurons.
Neurons are very fragile. You must be extremely cautious and gentle when approaching and pressure cycling the cells and retracting the pipette tip to causing serious damage. After electroporation, the cells can be subjected to various experimental analysis, including electrophysiology and imaging.
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