These methods were designed for use with retinal ganglion cells (RGCs) from Long-Evans rats. All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO), institutional, federal, and state guidelines regarding animal research.
Cell culture reagents were obtained from Gibco (Grand Island, NY). The fluorescent tracers 4′, 6-diamidino-2-phenylindole (DAPI), 1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindocarbocyanine (DiI), and 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-ASP) were obtained from Molecular Probes (Eugene, OR). Hank’s balanced salt solution (HBSS) was obtained from BioWhittaker, Inc. (Walkersville, MD). Chambered #1.0 German borosilicate coverglasses were obtained from Fisher Scientific (Pittsburgh, PA). Papain was obtained from Worthington Biochemical (Freehold, NJ). Staurosporine was obtained from Alexis Biochemical (San Diego, CA). Unless noted, all other reagents were obtained from Sigma (St. Louis, MO).
Retinal ganglion cell labeling and culture
Retinal ganglion cells were labeled and cultured using modifications of previously described methods (Levin et al., 1996
). RGCs were retrogradely labeled by stereotactic injection of the fluorescent tracer DAPI (5 mM) dissolved in dimethylformamide into the superior colliculi of anesthetized postnatal day 2–4 Long-Evans rats. To do this, incisions were made across the top of their heads to reveal bregma. The two points of injection were 2 mm rostral and 2 mm lateral from the center of bregma. With a 10 μL Hamilton syringe, 4 μL of tracer solution was delivered at each injection site at a depth of 4 mm. The wound was sealed with cyanoacrylate adhesive and bandaged with a small strip of paper towel and cyanoacrylate.
To study labeling efficiency, DiI was coinjected with DAPI. Tracer solutions consisted of 5.4 mM DiI and 2.5 mM DAPI in dimethylformamide, and 4 μL of solution was delivered to each injection site. During the 3–4 days after injection, the dyes travel by retrograde transport from RGC projection sites in the superior colliculi to the RGC somas in the retina. DiI uniformly labels neurons via lateral diffusion in the plasma membrane, while DAPI stains nuclei specifically with little or no cytoplasmic labeling.
At postnatal day 7–8, the animals were sacrificed by decapitation, the eyes were enucleated, and the retinas were dissected free in HBSS. After two incubations in HBSS containing papain (12.5 U/ml), each for 7 minutes at 37°C, the retinas were gently triturated with a serological pipette and plated on poly-L-lysine-coated chambered coverglasses at a density of approximately 2000 cells/mm2. The cells were cultured for 24 hours in Neurobasal A (Gibco) with 0.7% methyl cellulose, 2% B27 serum-free supplement (Gibco), and gentamicin (5 μg/ml) at 37°C and 5% CO2.
Additional studies of labeling efficiency were performed by injecting 6 μL of a tracer solution containing 2.5 mM DAPI and 10 mg/ml 4-Di-10-ASP in 50% DMSO/50% H2O in the manner described above. Animals were sacrificed one week later, and the eyes enucleated in HBSS. Retinas were removed, wholemounted, and imaged immediately under appropriate filters.
Epifluorescence microscopy for visual assessment
DAPI-positive cells were identified using a Zeiss Axiovert 135 inverted microscope and filters for DAPI (330 nm excitation, 450 nm emission). DiI positive cells were identified with appropriate filters (540 nm excitation, 610 nm emission). Images of the cells were acquired with a cooled CCD camera (Roper Scientific, Trenton, NJ) mounted on the microscope, using Metafluor software at a binning of 1, an exposure time of 200 ms, and 1X gain. For the DAPI-DiI dual injection condition, cells were scored as DAPI−/DiI−, DAPI+/DiI−, DAPI−/DiI+, or DAPI+/DiI+.
Retinal wholemounts were imaged with a digital camera (Axiocam HRc) attached to a fluorescence microscope (Axiophot; Carl Zeiss Meditec, Inc., Dublin, CA) at several levels of magnification. Cells were identified in the respective images as being DAPI+ and 4-Di-10-ASP+ before merging of the pictures to check for colocalization of staining with the two tracers. In separate experiments (n = 4), staurosporine was added to the culture medium to induces apoptosis by inhibiting protein kinases and prevent ATP binding. Following cell plating, staurosporine prepared in DMSO was added to a final concentration of 1 μM (0.1% DMSO). Images of DAPI positive cells were acquired 24 hours later.