A primary neuron culture system for functional studies of anoxia tolerance in turtles

Cultured neuronal models for non-mammalian vertebrates are uncommon but could prove useful for investigating mechanisms of exceptional physiological performance, such as anoxia tolerance. We describe a procedure to isolate, culture and characterize cerebrocortical neurons of extremely anoxia-tolerant painted turtles; we then imposed anoxia while recording reactive oxygen species (ROS) using fluorescence photometry. Cerebrocortical sheets from hatchlings were dissociated enzymatically and mechanically, and cultured for ≤7 days. Within 24 h, most cells possessed classic neuronal morphology with identifiable soma and fine neurites. Immunocytochemistry showed MAP2-positive cells accounted for 90.9% of DAPI-positive cells. Ca2+ recordings (Fura-2) demonstrated neurons were immediately excitable with KCl and glutamate, but not acetylcholine. ROS recordings (CM-H2DCFDA) showed they avoided excessive ROS production post-anoxia, like other turtle brain preparations, but with higher signal and temporal resolution. These approaches should extend previous work in other brain preparations to isolated cells that possess the morphological and functional features of neurons.