Isolation and culture of adult rat hippocampal neurons

Inability to culture adult central neurons and the failure of injured neurons to regenerate in the brain could be due to genetic controls or environmental inhibitors. We tested the environmental inhibitor hypothesis by attempting to regenerate adult rat neurons in B27/Neurobasal culture medium, a medium optimized for survival of embryonic neurons. To isolate neurons from their numerous connections, papain was the best of six different proteases screened on slices of hippocampus for survival of isolated cells after 4 days of culture. Use of a density gradient enabled separation of oligodendroglia and some enrichment of neurons and microglia from considerable debris which was inhibitory to sprouting and viability. With these techniques, about 900000 viable neurons were isolated from each hippocampus of any age rat from birth to 24-36 months, near the median mortality. FGF2 was found to enhance viability at least 3-fold to 40-80%, independent of age, without affecting the length of the processes. Neurons were cultured for more than 3 weeks. These methods demonstrate that hippocampal neurons can regenerate axons and dendrites if provided with adequate nutrition and if inhibitors are removed. They also will enable aging studies. Therefore, the concept of environmental growth restriction may be more appropriate for neurons in the brain than the concept of a genetic block that precludes regeneration of processes.