In light of the importance of the calcium ion in neuronal function, the object of this proposal is, first of all, to obtain a better understanding of the control of intraneuronal calcium by studying the functional significance of the 28,000 Mr brain calcium binding protein (calbindin). The calbindin protein will be introduced into single cells using a patch pipette and we will determine possible effects on intracellular calcium and changes in calcium channel activity. Using CA1 hippocampal cells we will also determine whether the presence of calbindin can protect against cell death which results after exposure to high levels of glutamate and subsequent excessive increase in intracellular calcium. In addition, we will transfect PC12 cells with the calbindin gene and we will determine if the presence of calbindin has an effect on neurite outgrowth which occurs in response to nerve growth factor (NGF). If changes are observed, studies using site directed mutagenesis will allow the elucidation of structure-function relationships. We also propose to study the regulation of calbindin in cultured, dissociated neurons. Preliminary findings concerning an induction of calbindin by NGF in a dispersed culture of rat cerebellar cells will be further investigated. In addition, we will examine the possibility of regulation of calbindin expression by calcium. Our second objective will be to determine whether there are alterations in other calcium related genes, such as calcium calmodulin dependent protein kinase (CaM kinase II), with seizure activity. Changes in CaM kinase II gene expression will be examined in hippocampus, cortex and subcortical structures (including amygdala, substantia nigra and striatum) of commissure and amygdaloid kindled rats. In addition, changes in CaM kinase II gene expression will also be examined in these structures after pentylenetetrazole induced seizures. Finally, in order to obtain the best understanding of the molecular basis of epileptogenesis, we will determine by differential hybridization which genes are altered at early times after kindling induced seizures (1 hour or 24 hours) or 28 days after the last kindled seizure. Changes in gene expression in hippocampus and substantia nigra will be initially investigated. DNA sequences of clones obtained by differential hybridization will be compared to known sequences. cDNAs of genes altered either at early times or 28 days after the last kindled seizure will be used to determine by Northern analysis whether changes in these genes are also observed with chemically induced seizures. We also propose to examine human brain tissue from epileptic patients for changes in these genes. These studies should enable us to identify alterations in specific genes involved in convulsive neuronal activity. It is likely from these studies that important advances can be made concerning the molecular level changes underlying epileptogenesis.
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