Microinjections were executed using previously described methods (75, 78). A Nikon Eclipse FN1 upright microscope with a 10× objective and a 40× water objective was placed on an air table. The tissue chamber used was assembled in the laboratory and consisted of a plastic base (50 mm × 75 mm) with a petri dish (60 mm × 10 mm) epoxied to the base. A platinum wire was attached so that the ground wire could be connected to the bath by an alligator clip. The negative terminal of the electric current source was connected to a glass micropipette filled with 2 μl of 8% Lucifer yellow dye (Thermo Fisher Scientific, catalog no. L453). Micropipettes (A-M Systems, catalog no. 603500) with highly tapered tips were pulled fresh the day of use. A manual micromanipulator was secured on the air table with magnets that provided a 45° angle for injection. Brain slices were placed into a small petri dish containing 1× PBS and DAPI for 5 min at room temperature. After incubation in DAPI, slices were placed on dental wax, and then, a piece of filter paper was used to adhere the tissue. The filter paper was then transferred to the tissue chamber filled with 1× PBS and weighted down for stability. The 10× objective was used to visualize advancement of the tip of the micropipette in XY and Z until the tip was just a few micrometers above the tissue. The 40× objective was then used while advancing the tip into the CA1 region of the hippocampus. Once the microelectrode contacted a neuron, 2 nA of negative current was used for 5 min to fill the neuron with Lucifer yellow. After 5 min, the current was turned off, and the micropipette was removed from the neuron. Neuron impalement within the CA1 occurs randomly in a blind manner. If the entire neuron does not fill with dye after penetration, then the electrode is removed and the neuron is not used for analysis. Multiple neurons were injected in each hemisphere of the hippocampus of each animal. After injection, the filter paper containing the tissue was moved back into the chamber containing 1× PBS. The tissue was carefully lifted off the paper and placed on a glass slide with two 125-μm spacers (Electron Microscopy Sciences, catalog no. 70327-20S). Excess PBS was carefully removed with a Kimwipe, and the tissue was air-dried for 1 min. One drop of Vectashield (Vector Labs, catalog no. H1000) was added directly to the slice; the coverslip (Warner, catalog no. 64-0716) was added and sealed with nail polish. Injected tissue was stored at 4°C in the dark.

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