Intracellular injection of astrocytes in lightly fixed tissue slices was performed as previously described, with some modifications (Buhl et al., [1990], PMID: 2262598). The brain was placed in ice-cold PBS and cut into coronal slices with a vibratome at a thickness of 100 µm, and stored in PBS at 4°C until used. The slices were placed under a 60x water objective (NA 1.4) and observed with an Olympus BX50WI microscope using infrared-DIC optics (Olympus, Melville, NY). Astrocytes in the upper blade of the dentate gyrus were identified by the shape and size of their somata. Glass micropipettes (OD 1.00 mm, ID 0.58 mm; resistances 100-400 M) were pulled on a vertical puller (David Kopf Instruments, Tujunga, CA) and backfilled with either 5% aqueous LY in 200 mM KCl. Astrocytes were impaled and iontophoretically injected with dye using 1-second pulses of negative current (0.5 Hz) for 1-2 minutes. After several cells were filled, the slices were placed in ice-cold 4% PFA for at least 1 hour. The slices were then ready to be immunolabeled. The rabbit anti-EphA4 antibody was provided by Dr. Elena Pasquale (The Burnham Institute, La Jolla, CA). EphA4 was subsequently detected using goat anti-rabbit Cy5. Slices were coverslipped using Gelvatol (Harlow and Lane, [1988]) and allowed to set overnight at room temperature before they were examined. Image acquisition and analysis: Specimens were examined using a Radiance2000 laser scanning confocal system (Bio-Rad, Hercules, CA) attached to a Nikon E600FN microscope (Kanagawa, Japan). A 60x oil immersion (NA 1.4) objective was used to image LY-filled astrocytes.