Hypoxia-induced alterations in membrane properties and pH of rat dorsal vagal motoneurones

The studies presented in this thesis have focused on the immediate effects of hypoxia on the membrane properties of dorsal vagal motoneurones (DVMs). There were two main objectives: firstly, to determine the ionic basis of the hypoxia-induced effects on membrane potential and on the characteristics of the action potential and secondly, to determine whether hypoxia-induced changes in pH could be responsible for the observed changes in membrane potential. Membrane potential was unaffected by hypoxia in 11 % of 111 DVMs. An hyperpolarisation accompanied by a decrease in input resistance occurred in 44% of DVMs; the remaining 45% depolarised with either an increase (60%) or decrease in input resistance ( 40% ). These responses did not reflect action potential dependent release of neurotransmitter because they were not affected by 0.3-1 μM TIX. The ionic basis of the hypoxia-induced changes in membrane potential was established by use of various ion channel blockers. The results suggested that hypoxia enhances both IK(DR) and an inward current whose identity remains to be determined, but suppresses IA. In DVMs that hyperpolaiise during hypoxia, the increase in IK(DR) outweighs the increase in the inward current and the decrease in IA. In depolai·ising DVMs the decrease in IA and increase in the inward current outweigh the increase in IK(DR). The change in input resistance is determined _by the relative sizes of current enhancement or suppression. In addition, there is an hypoxia-induced enhancement of a Ca2+-mediated component of the action potential and inhibition of the IK(Ca) that underlies the long AHP of DVMs. The specificity of some of the ion channel blockers which had been used was confirmed in a voltage-clamp study. This approach also revealed that the thresholds of activation of the voltage-dependent ion channels thought to be involved in the hypoxiaV induced membrane potential response were in the vicinity of resting membrane potential. In addition, the voltage-clamp studies revealed that hypoxia enhances a voltage-independent or "leak" current. In most neurones this response could be blocked by carbachol. In certain drug combinations (particularly in the presence of Ca2+ and K+ channel blockers) hypoxia induced a rapid depolarisation or rapid inward current. This appeared similar to "rapid anoxic depolarisation" or "spreading depression-like hypoxic depolarisation". However, this type of response was never observed during hypoxia in standard ACSF. The effects of hypoxia on various voltage-dependent ion channels raised the question of how hypoxia modulates ion channel behaviour. Therefore, experiments were designed to explore the hypothesis that hypoxia-induced decreases in pHi or pH0 alter the properties of ion channels. Direct measurements of pHi and pH0 revealed that resting pHi of DVMs maintained in vitro is quite variable. In hypoxia, changes in pHi and pH0 were generally found to precede changes in membrane potential. Furthermore, the directions and amplitudes of the hypoxia-induced changes in membrane potential were found to be highly correlated with prehypoxic values of pHi, This same relationship occurred in hypoxic 25 mM HEPES ACSF. In hypoxic 10 mM HEPES ACSF changes in membrane potential were independent of resting pHi; the greater extracellular acidity appeared to be responsible for the different membrane potential response. Further studies of the effects of 10 mM HEPES buffer revealed that changing from oxygenated bicarbonate/CO2 ACSF to oxygenate~ 10 mM HEPES ACSF altered the nature of the ion currents present at resting membrane potential. IA, was abolished and there was a reduction of a Mn2+ sensitive current, most likely the T-type lea, As a result IA did not contribute to the hypoxia-induced depolarisation. Rather, the depolarisation was attributed to an increase in T-type lea and a decrease in an as yet unidentified current, although a small increase in IK(DR) opposed these effects. r VI The experiments described in this thesis suggest that resting pHi in DVMs determines which voltage-dependent ion channels are open at resting membrane potential. It was hypothesised that pH modulates ion channels by affecting the voltagedependence of their activation or inactivation. Thus, for a transient current, pHi may determine the size of the window current at resting membrane potential. This would then, in part, determine the effects of hypoxia-induced changes in pHi or pH0 on membrane potential by determining which voltage-dependent channels are available to be opened or closed.