Hypoxic-ischemic encephalopathy is an important cause of long-term neurological morbidity in babies, children, and adults. How vulnerable the brain is to hypoxic-ischemic injury depends critically on postnatal maturation. Activation of neuronal glutamate receptors plays a major role in the excitotoxic neurodegeneration that characterizes acute brain injury from hypoxia-ischemia. The importance of postnatal maturation in determining vulnerability to hypoxia-ischemia highlights the need for a precise understanding of how postnatal maturation regulates the intracellular components of excitotoxicity. The major objective of this application is to characterize the precise developmental regulation of the calcium-dependent processes that lead to excitotoxic neurodegeneration. To achieve this objective, the following Specific Aims are proposed: (i) Identify how plasma membrane Na+/Ca2+ exchange, ER Ca2+ sequestration and mitochondrial Ca2+ sequestration change with postnatal development. Hippocampal neurons cultured from P 0-5, P 10-15, and P 18-25 rats will be exposed to a step increase in intracellular [Ca2+], and the contributions of the mitochondria, ER, and plasma membrane Na+/Ca2+ exchanger to the return of intracellular [Ca2+] to baseline will be measured. (ii) Assess the importance of developmental changes to these homeostatic mechanisms in determining vulnerability to NMDA. NMDA-induced loss in viability will be measured in neuronal cultures from rats of the same age groups in the presence and absence of specific blockade of these mechanisms. (iii) Identify the role of mitochondrial Ca2+ accumulation in the increased dissipation of mitochondrial membrane potential that occurs during postnatal development during NMDA exposure. NMDA effects on delta psi will be measured in neurons from the same 3 age groups and correlated with changes in mitochondrial [Ca2+], and cytosolic [Ca2+] in the presence and absence of mitochondrial Na+/Ca2+ exchange blockade. Isolated uniporter and Na+/Ca2+ exchanger activity will be measured in permeabilized neurons from P 0-5 and P 18-25 rats. (iv) Determine the extent to which alterations in mitochondrial Ca2+ accumulation contribute to the postnatal development of excitotoxic vulnerability. NMDA induced neuronal death will be measured in the presence and absence of blockade of the Na+/Ca2+ exchanger and of the mitochondrial transition pore.
