The aim of the proposed experiments is to identify the pattern of distribution of creatine kinase activity in neurons. Specifically, the study will measure the activity of creatine kinase in pre- and post-synaptic elements in the dentate gyrus of the rat. The contribution of mitochondrial and cytoplasmic isozymes will be measured for each element. This will be done by measuring creatine kinase activity by enzymatically coupled fluorometric assays of microdissected tissue from three groups: 1) Control rats, 2) Rats which have stereotaxic lesions of the entorhinal cortex to decrease presynaptic elements, and 3) Rats which have colchicine-induced lesions of the dentate granule cells to reduce post-synaptic elements. Three discrete regions will be assayed for each group: 1) The cell body layer of the dentate granule cells, 2) The medial 1/3 of the molecular layer of the dentate gyrus, and 3) The distal 1/3 of the molecular layer. Regions 2 and 3 contain the synapses between neurons of the entorhinal cortex and dentate granule cells. The contribution of mitochondrial and cytoplasmic isozymes in each region will be measured by first separating the isozymes from pooled samples by electrophoresis. The intracellular localization of the cytoplasmic isozyme (BB-CK) will be further characterized by application of antibody specific for BB-CK. The results will indicate if any one structure in the dentate gyrus (i.e. soma, dendrite, axon, axon terminal) contains elevated creatine kinase activity. The results will further determine if BB-CK is preferentially localized to intracellular structures, such as the cell membrane of dendritic spines. Previous studies showed that augmenting the phosphocreatine content of the dentate gyrus prolongs neuronal function during anoxia by maintaining ATP levels. Therefore, a site with elevated BB-CK activity is likely to be the location of synaptic transmission failure during anoxia. Locating such a site will greatly assist in determining the mechanism by which anoxia produces electrical quiescence and may suggest a means of prolonging neuronal function and viability during energy deprivation.
| Whittingham, T S; Warman, E; Assaf, H et al. (1989) Manipulating the intracellular environment of hippocampal slices: pH and high-energy phosphates. J Neurosci Methods 28:83-91 |
