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Mechanisms of anoxia tolerance in the turtle cortex Doll, Christopher Joseph


The high sensitivity of the mammalian brain and the insensitivity of the turtle brain to O₂ deprivation led to the use of cortical slice preparations in both species being utilized for a comparative study of anoxia tolerance. To assess anoxic survival, intracellular recording techniques were employed. Turtle neurons survived both anoxia (aCSF equilibrated with 95% N2 /5 % CO₂) and pharmacological anoxia (anoxia + 1mM NaCN) for 180 min. with no measurable degradation. Rat pyramidal neurons responded with a decrease in whole cell resistance followed by transient hyperpolarization and a subsequent depolarization to a zero membrane potential (41.3 ± 6.5 min., anoxia; 25.8 ± 12.6 min., pharmacological anoxia). Pharmacological ischemia (pharmacological anoxia + iodoacetate 10 mM) caused a rapid decrease in whole cell resistance, transient hyperpolarization, and a rapid depolarization in both turtle (4.6 ± 1.1 min.) and rat (3.1 + 0.5 min.) neurons. Ouabain perfusion caused a rapid depolarization in the rat cortical neuron (8.6 ± 1.1 min.), but no initial decrease in whole cell resistance or a hyperpolanzation. Calorimetric measures converted to ATP utilization rate indicated that the turtle cortical slice has an initial ATP utilization of 1.72 μmoles ATP/g/min. which agrees closely to in vivo whole brain metabolic measures. This value supports a 9 fold lower metabolic rate compared to analogous guinea pig cortical slice preparations. Based on heat depression measures, resulting ATP utilization estimates indicated a metabolic depression of 30 % (nitrogen) and 42% (pharmacological anoxia). Heat flux changes over pharmacological anoxia, support a large initial Pasteur effect which gradually declines over the 120 min. insult interval. Activities of hexokinase and lactate dehydrogenase were similar between the rat and turtle cortical slice (25 °C), but the turtle cortex only expressed 80 % of the activity of the rat cortex for citrate synthase. Surprisingly, the turtle cortical slice did not exhibit a change in any measured adenylate parameter up to 120 min. of anoxia or pharmacological anoxia. Significant changes did occur in [ADP], ATP/ADP ratio, and energy charge at 240 min. In order to assess difference in ion leakage in both the turtle and rat pyramidal neurons, intracellular recording techniques for short term anoxia (120 min.) and whole cell patch clamp techniques (on cell populations) for long term anoxia (6 -9 hrs.) were utilized. Both techniques indicated that turtle cortical pyramidal cells did not change in conductance (whole cell conductance or specific membrane conductance) with anoxia. Whole cell patch clamp techniques supported a 4.2 fold higher specific membrane conductance in rat pyramidal neurons compared to turtle neurons at the same temperature (25 °C) which was accentuated by temperature so that rat pyramidal neurons at 37°C were 22 times more conductive than turtle neurons at 15°C. A conductance Q₁₀ of 1.9 was measured for both turtle (15-25°C) and rat (25-35°C) pyramidal neurons. To asses pumping activity capacity, Na⁺-K⁺ ATPase activity was measured in cortical slices of both species. At the same temperature (25 °C) a 2.3 fold higher activity was measured in the rat cortex compared to the turtle supporting the patch clamp results of a lower normoxic specific membrane conductance in the turtle cortex. Taken together these results support that the turtle brain is able to survive anoxia through an enhanced glycolytic capability, a low normoxic brain metabolism with the ability to further depress metabolism during anoxia. Electrophysiological techniques support reduced ion pumping through reduced ion leakage as one mechanism for a depressed normoxic metabolic rate in the turtle cortical slice but do not support further down regulation of channel activity with anoxia.

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