Ion Transport and Membrane Potential in CNS Myelinated Axons II. Effects of Metabolic Inhibition

Abstract

Leppanen, Lisa and Peter K. Stys. Ion transport and membrane potential in CNS myelinated axons. II. Effects of metabolic inhibition. J. Neurophysiol. 78: 2095–2107, 1997. Compound resting membrane potential was recorded by the grease gap technique (37°C) during glycolytic inhibition and chemical anoxia in myelinated axons of rat optic nerve. The average potential recorded under control conditions (no inhibitors) was −47 ± 3 (SD) mV and was stable for 2–3 h. Zero glucose (replacement with sucrose) depolarized the nerve in a monotonic fashion to 55 ± 10% of control after 60 min. In contrast, glycolytic inhibition with deoxyglucose (10 mM, glucose omitted) or iodoacetate (1 mM) evoked a characteristic voltage trajectory consisting of four distinct phases. A distinct early hyperpolarizing response ( phase 1) was followed by a rapid depolarization ( phase 2). Phase 2 was interrupted by a second late hyperpolarizing response ( phase 3), which led to an abrupt reduction in the rate of potential change, causing nerves to then depolarize gradually ( phase 4) to 75 ± 9% and 55 ± 6% of control after 60 min, in deoxyglucose and iodoacetate, respectively. Pyruvate (10 mM) completely prevented iodoacetate-induced depolarization. Effects of glycolytic inhibitors were delayed by 20–30 min, possibly due to continued, temporary oxidative phosphorylation using alternate substrates through the tricarboxylic acid cycle. Chemical anoxia (CN− 2 mM) immediately depolarized nerves, and phase 1 was never observed. However a small inflection in the voltage trajectory was typical after ≈10 min. This was followed by a slow depolarization to 34 ± 4% of control resting potential after 60 min of CN−. Addition of ouabain (1 mM) to CN−-treated nerves caused an additional depolarization, indicating a minor glycolytic contribution to the Na+-K+-ATPase, which is fueled preferentially by ATP derived from oxidative phosphorylation. Phases 1 and 3 during iodoacetate exposure were diminished under nominally zero Ca2+ conditions and abolished with the addition of the Ca2+ chelator ethylene glycol-bis(β-aminoethyl ether)- N,N,N′,N′-tetraacetic acid (EGTA; 5 mM). Tetraethylammonium chloride (20 mM) also reduced phase 1 and eliminated phase 3. The inflection observed with CN− was eliminated during exposure to zero-Ca2+/EGTA. A Ca2+-activated K+ conductance may be responsible for the observed hyperpolarizing inflections. Block of Na+ channels with tetrodotoxin (TTX; 1 μM) or replacement of Na+ with the impermeant cation choline significantly reduced depolarization during glycolytic inhibition with iodoacetate or chemical anoxia. The potential-sparing effects of TTX were less than those of choline-substituted perfusate, suggesting additional, TTX-insensitive Na+ influx pathways in metabolically compromised axons. The local anesthetics, procaine (1 mM) and QX-314 (300 μM), had similar effects to TTX. Taken together, the rate and extent of depolarization of metabolically compromised axons is dependent on external Na+. The Ca2+-dependent hyperpolarizing phases and reduction in rate of depolarization at later times may reflect intrinsic mechanisms designed to limit axonal injury during anoxia/ischemia.