Imaging Mitochondrial Function in Excitotoxicity

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DESCRIPTION (provided by applicant): Compared to most other organ systems, normal brain function requires a disproportionately large energy supply, and even transient disruption of brain metabolism can contribute to catastrophic loss of cognitive or motor function in a wide range of neurodegenerative disorders. Ischemic insults can lead to unregulated release of the neurotransmitter glutamate, and lead to inappropriate over excitation of neurons to the point of triggering cell death. The process of cell damage following excessive glutamate receptor activation has been termed "excitotoxicity", and may also be involved in a range of disorders including seizure activity, Parkinson's Disease and ALS. Strategies that maintain appropriate metabolic function may be a critical consideration for the design of future therapeutic interventions for excitotoxic injuries. The success of such interventions relies on understanding metabolic demands involved in different types of glutamate excitotoxicity. Experiments in this proposal will evaluate mitochondrial function in acute hippocampal slices, to evaluate the mechanisms involved in mitochondrial function changes in situ, following glutamate receptor stimulation. A major approach used to study mitochondrial function will be fluorescence imaging of intrinsic metabolic signals, an approach which has been validated in many biochemical and some imaging studies, but which has received a resurgence of interest because of the application of high resolution imaging to intact preparations. The use of imaging approaches in acute slices allows the contributions of glial and neuron metabolism to be differentiated in intact preparations. Responses to endogenously-released glutamate (either during electrical depolarization or hypoxic/hypoglycemic challenges) to be compared with responses to glutamate receptor subtype-selective agonists. Single- and 2-photon imaging will be used to identify cellular sources of mitochondrial signals, single cell electrophysiology/imaging to identify mechanisms and cells responsible for metabolic changes and pharmacological interventions that selectively modify metabolic pathways responses in neurons vs. glia. Intrinsic fluorescence studies will be complemented by fluorescence imaging of mitochondrial inner membrane potential, and single cell electrophysiological analysis of ionic fluxes contributing to metabolic dysfunction. Hippocampal CA1 neurons will be the subject of most studies, because of their sensitivity to excitotoxic damage and the extensive literature on mechanisms of hippocampal pyramidal neuron physiology and mechanisms of excitotoxic cell death. For studies of mitochondrial function in neurons destined to die following transient ischemia (Specific Aim 3), we will utilize preparations from gerbils subjected to transient forebrain ischemia.