A method for labeling embryonic rat medial septal region projection neurons, in vitro, using fluorescent tracers

A retrograde labeling method is described in which rat embryonic (E18, E21) and postnatal (P1) medial septal neurons were labeled with succinyl wheat germ agglutin-fluorescein, fluorescent green microspheres, or 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (Dil) following in vitro hippocampal injections. The brains were removed and immediately immersed in oxygenated Tyrode solution. Dye was pressure injected into the hippocampus bilaterally. After incubating the brain in oxygenated Tyrode, the medial septal region was removed. The neurons were dissociated and cultured at medium density in 35 mm dishes with a hole in the bottom covered by a coverslip with a grid. The neurons were observed with a low light system, and cell counts were made at 5, 24, and 48 h. Labeled and unlabeled neurons showed considerable neurite outgrowth and acetylcholinesterase activity in culture. Highly reproducible labeling was obtained, with Dil giving the best results. Dil labeled the neurons in vitro, was retained during culture for 1 week, and was compatible with cell survival.