Experiments were performed on a patch electrophysiology rig under an Olympus microscope with differential interference contrast optics and immersion lenses. Pipettes were back loaded with plasmid DNA, fluorescent dextran (tetramethylrhodamine‐ or fluoroscein‐conjugated dextran (3000 MW, Invitrogen # D3307 and D3305, respectively, 1–3% in 0.9% NaCl), or neurobiotin (1–2% in 0.9% NaCl) and mounted on the recording headstage. Brain slices were placed in the recording chamber and perfused at 1–2 mL per minute. The pipette tip was guided onto the surface of the target cell until a dimple was formed. Pipette patency was maintained with positive pressure as required. Following electroporation the pipette was carefully retracted from the cell and reused until clogging occurred.
The same approach was used for transfection of neurons in organotypic culture, except pipettes were filled with freshly filtered plasmid DNA in 0.9% NaCl (0.3–3 μg/μL). SCE of organotypic cultures was typically completed within 15 min. Transfected neurons were washed in fresh media and restored to the incubator for 24–48 h before fixation and imaging of reporter‐expressing neurons.
The same approach was used for transfection of neurons in organotypic culture, except pipettes were filled with freshly filtered plasmid DNA in 0.9% NaCl (0.3–3 μg/μL). SCE of organotypic cultures was typically completed within 15 min. Transfected neurons were washed in fresh media and restored to the incubator for 24–48 h before fixation and imaging of reporter‐expressing neurons.