Neuronal activity and energy metabolism are tightly coupled processes. The critical dependence of the brain on oxidative metabolism forms the basis for the use of an important energy-deriving enzyme, cytochrome oxidase (CO), as a functional metabolic marker of neurons. Studies in the past decade have demonstrated that the activity of this enzyme rises and falls with neuronal activity, and that regulation occurs at the regional, laminar, cellular, and subcellular levels. The molecular mechanisms of CO activity regulation is unknown. Experiments outlined in this proposal will examine protein levels and mRNA levels as possible sites of CO activity regulation in the CNS. Our working hypotheses are: (1) The adjustment of CO activity in response to normal or altered functional demands is due to an adjustment in CO protein levels; (2) CO protein levels, and any adjustment of it, are directly related to CO mRNA levels; and (3) There is coordinated regulation of mitochrondrial- and nuclear-derived CO subunits as well as of the mRNAs from the two genomic sources under normal and functionally inactivated states. We propose to test these hypotheses in the rodent barrel fields and the primate lateral geniculate nucleus by three parallel approaches; histochemistry, immunohistochemistry, and in situ hybridization. These approaches offer the distinct advantage of precise cellular localization and cross comparisons in adjacent sections. Double-labeling of CO histochemistry and CO immunohistochemistry, as well as double immunolabeling of CO and GABA in the same section will also be done for the identification of geniculate interneurons (GABAergic). The immunohistochemical probes will be polyclonal antibodies monospecific against brain cytochrome oxidase subunits. For in situ hybridization, riboprobes and synthetic oligodeoxynucleotides will be generated against known sequences of mitochrondrial and nuclear genes for CO subunits. The distribution of relative levels of CO activity, protein, and mRNAs will be compared in barrel fields and lateral geniculate neurons under normal and sensory deprived conditions. Data obtained may help us gain better understanding of the cellular and molecular bases of the dynamic neuronal responses to normal and altered states of functioning.
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