Anoxia tolerance in isolated neurons from the Western painted turtle: insights from mitochondrial energetics

The Western painted turtle ( Chrysemys picta bellii ) is the only known tetrapod to survive anoxia for more than 170 days at 3°C. Among the most important mechanisms of survival in the turtle brain are spike and channel arrest, which greatly decreases ATP consumption and neuronal activity via mitochondrial suppression of ROS generation and calcium release. In mammalian brain, however, oxygen deprivation leads to excitotoxicity and cell death within minutes. Comparatively, reptiles have lower mitochondrial density and proton leak resulting in tighter oxygen coupling. Additionally, previous research in cortical sheets has shown mild mitochondrial membrane potential (ΔΨm) depolarization preserving the proton electrochemical gradient and avoiding membrane collapse along with inhibition of mitochondrial ROS signaling GABA release suppressing electrical activity. Turtles have also been shown to have a well-developed antioxidant system. Lastly, we are unaware of studies measuring changes in NAD(P)H during anoxia, although it is expected that the NADH/NAD+ ratio will be maintained to prevent ROS formation. Using isolated turtle cortical neurons, we tested the hypothesis that anoxia would (1) slightly depolarize ΔΨm, (2) NAD(P)H pool would increase, and (3) ROS production will be limited. Neurons were dissociated from the cerebrocortex using enzymatic digestion and mechanical trituration and cultured for up to 7 days on glass coverslips. Rhodamine-123 was used to measure ΔΨm, NAD(P)H autofluorescence was used to measure redox state changes and glutathione peroxidase (GPX) antioxidant responses, NAD(P)H autofluorescence, and CM-H2DCFDA was used to measure ROS production. During anoxia, Ψm hyperpolarized, indicating dysregulation of ion exchangers and reversal of ETC, while NAD(P)H decreased and was reversed by NADPH-oxidase (NOX) inhibition with apocynin. NADPH acts as a substrate for NOX to produce ROS in addition to being a substrate for antioxidant systems. Surprisingly, ROS production only showed a small increase during reoxygenation, but not during normoxia-anoxia transition indicating that, similarly to previous cortical slices measurements, isolated painted turtle neurons avoid excessive ROS production. Control experiments with pyrogallol showed a large increase in ROS. Upon inhibition of glutathione reductase (GR) enzyme, NAD(P)H levels increased both during normoxia and anoxia, indicating that GPX is a constitutive process in painted turtle neurons and that other antioxidant systems can compensate for the loss of GR. All together these findings indicate: 1) constitutive activities of GR, in conjunction with other antioxidant systems, are adequate to prevent anoxia-induced increases in ROS, 2) ΔΨm and cellular redox state have no bearing on ROS production in turtle neurons, and 3) there is some level of NADPH-oxidase activity during anoxia, but it is unclear whether that activity is induced, or if it is constitutive. National Sciences Foundation[1253939 to D.W.]; and the Saint Louis University President’s Research Fund [to D.W.] This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.