| Summary: | 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. === Science, Faculty of === Zoology, Department of === Graduate
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