In the past several years, one main focus of the investigator's laboratory has been to examine the response of central neurons in a number of brain regions to O2 deprivation. The rationale for this effort has been to try to understand how neuronal excitability is altered and the nature of the cascade of biologic and physiologic events that occur during anoxia in nerve cells. The investigators are particularly interested in the mechanisms that can lead to neuronal injury and those that, when activated, can prevent or delay injury. Recently, the investigators' have made interesting observations regarding membrane ionic events that link metabolism to excitability. One of these observations pertains to the voltage-sensitive Na+ channels in central neurons. An early event during anoxia in mature (adult) neocortical neurons seems to be a profound inhibition of the steady-state availability of these channels with a major reduction in Na+ current (INa) and a decrease in neuronal excitability. This observation is important because this may be an adaptive strategy in the adult in that the decrease in excitability will lessen O2 consumption and minimize the mismatch between demand and supply. The investigators are not sure how the neonatal neocortical cells will respond in terms of INa. The investigators therefore focus in this proposal on the study of the Na+ current in neocortical neurons during graded hypoxia and examine 3 separate hypotheses: 1) Neocortical neurons decrease their excitability during graded hypoxia by inhibiting INa in a graded manner in the adult but not in the neonate; 2) the alterations in INa kinetics during O2 deprivation in neocortical neurons are due to changes in specific cytosolic factors and these are more pronounced in the adult than in the neonate; and 3) O2 deprivation alters INa via mechanisms that are either membrane-delimited or dependent on phosphorylation. These experiments will involve the use of the in vitro slice and microelectrode technique as well as patch clamp with whole- cell and single channel recordings in freshly dissociated neurons. Optical measurements of Ca++ i and H+ i using confocal microscopy will also be performed. All techniques are available and routinely performed in the principal investigator's laboratory. The investigators long term view and efforts are focussed on understanding the events that occur during anoxia in central neurons so that the investigators can manipulate cell behavior and possibly render mammalian neurons more tolerant to lack of O2. This will have major implications on a vast number of disease or conditions that span the age spectrum from the fetus to old age.
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